The most widely used reagent for fixation in histology is formaldehyde.
Fixation is a critical step in preparing biological tissues for microscopic examination, preventing degradation and preserving cellular structures. Formaldehyde serves as an exceptionally effective chemical fixative due to its ability to preserve tissue architecture and cellular components.
Understanding Formaldehyde and Formalin
Formaldehyde is a gas that is typically dissolved in water to create an aqueous solution known as formalin. This solution is the practical form used in laboratories.
- A 4% solution of formaldehyde is the standard concentration for many fixation protocols.
- This 4% formaldehyde solution is equivalent to a 10% solution of formalin. The term "10% formalin" is frequently used in histology and refers to a solution made by diluting 37-40% formaldehyde (the concentrated stock) 1:10 with water.
To enhance the stability of the formalin solution and prevent its polymerization into paraformaldehyde (a solid, ineffective form), approximately 10–15% methyl alcohol is often added. This stabilizer ensures the formaldehyde remains in its active, monomeric state, ready to react with tissue components.
How Formaldehyde Works
Formaldehyde acts primarily by cross-linking proteins within the tissue. This process involves forming chemical bonds between protein molecules, which:
- Stabilizes cellular structures: It hardens and stiffens the tissue, maintaining its original shape and preventing distortion.
- Inhibits enzymatic degradation: It inactivates endogenous enzymes (autolysis) that would otherwise break down the tissue.
- Prevents bacterial decomposition: It acts as an antimicrobial agent, preventing putrefaction.
Key Characteristics of Formaldehyde Fixation
| Feature | Description |
|---|---|
| Primary Action | Cross-links proteins, preserving cellular and tissue morphology. |
| Common Form | Formalin (aqueous solution of formaldehyde). |
| Standard Conc. | 4% formaldehyde (equivalent to 10% formalin). |
| Stabilizer | 10-15% methyl alcohol is added to prevent polymerization, ensuring solution stability and efficacy. |
| Advantages | Excellent tissue penetration, good preservation of general morphology, cost-effective, compatible with various staining techniques, and relatively stable. |
| Considerations | Requires proper ventilation and handling due to its irritant nature; can cause slight shrinkage or hardening of tissue over prolonged exposure. |
Practical Insights
- Preparation: Standard buffered formalin solutions are often used to maintain a stable pH, preventing the formation of formic acid which can cause pigment deposition in tissues.
- Time: The duration of fixation is crucial. Insufficient fixation leads to poor tissue preservation, while over-fixation can cause excessive hardening and interfere with subsequent processing or special staining.
- Safety: Always handle formaldehyde in a well-ventilated area, preferably under a fume hood, and use appropriate personal protective equipment (PPE) due to its irritant properties.
For more information on the broader field of tissue preparation, explore resources on histology techniques.
