What is Fixation?
Fixation is a crucial step in
histology that involves preserving biological tissues from decay and preparing them for staining and microscopic examination. This process helps maintain the structural integrity of the tissues and prevents autolysis and putrefaction.
Objectives of Fixation
The primary objectives of fixation include: Preserving tissue morphology and cellular structure
Preventing microbial contamination
Stabilizing cellular proteins and enzymes
Facilitating subsequent staining processes
Types of Fixatives
Fixatives can be broadly classified into two categories:
chemical and
physical fixatives. Chemical fixatives are further divided into two subcategories: aldehyde-based and oxidizing agents.
Chemical Fixatives
Aldehyde-Based Fixatives
Formaldehyde and glutaraldehyde are the most commonly used aldehyde-based fixatives. Formaldehyde, often used as formalin (a 10% solution of formaldehyde in water), is particularly effective for preserving tissue architecture. Glutaraldehyde is more efficient at cross-linking proteins and is preferred for electron microscopy.
Oxidizing Agents
Oxidizing agents like osmium tetroxide and potassium dichromate are used less frequently but are crucial for specific applications. Osmium tetroxide is primarily used for
electron microscopy due to its ability to preserve lipid membranes and provide excellent contrast.
Physical Methods
Physical fixation methods include freezing and microwave irradiation. These methods are advantageous because they can be rapid and reduce artifacts associated with chemical fixation.Freezing
Freezing is often used for preparing cryosections. Rapid freezing followed by sectioning with a cryostat preserves enzyme activity and antigenicity, making it suitable for
immunohistochemistry and enzyme histochemistry.
Microwave Irradiation
Microwave fixation is a relatively new technique that uses microwave energy to rapidly heat and fix tissues. This method can significantly reduce fixation time and improve the preservation of cellular structures.
Factors Influencing Fixation
The effectiveness of fixation depends on several factors: Temperature: Fixation is generally more effective at room temperature, but some tissues may require lower temperatures.
pH: The pH of the fixative solution can affect the preservation of cellular components. For example, formaldehyde works best at a neutral pH.
Fixative Concentration: The concentration of the fixative should be optimized to balance tissue penetration and preservation.
Fixation Time: The duration of fixation is critical. Under-fixation can lead to inadequate preservation, while over-fixation can cause excessive cross-linking and hardening of tissues.
Common Problems and Solutions
Several common issues can arise during fixation:Under-Fixation
Under-fixation can lead to poor tissue preservation and compromised structural integrity. This can be addressed by increasing fixation time or using a more concentrated fixative solution.
Over-Fixation
Over-fixation causes excessive cross-linking, making tissues hard and brittle. This can be mitigated by optimizing fixation time and using appropriate fixative concentrations.
Artifact Formation
Artifacts can result from improper fixation techniques, such as uneven penetration of the fixative. Ensuring thorough and even exposure to the fixative can help minimize artifacts.
Conclusion
Fixation is a vital step in histology that ensures the preservation and integrity of biological tissues for microscopic examination. Understanding the various fixation methods and their applications can significantly enhance the quality of histological studies. By carefully selecting and optimizing fixation protocols, researchers can obtain reliable and reproducible results.
