Tissue fixation is a fundamental step in preparing biological specimens for microscopic examination. This process involves treating tissues to preserve their structure, much like how pickling vegetables allows them to remain edible for extended periods. It is the initial stage in disciplines such as pathology and biological research, enabling scientists to study cellular and tissue architecture as it existed in the living organism and ensuring sample integrity for subsequent detailed analysis.
The Purpose of Preserving Tissue
The primary goal of preserving tissue is to halt the natural degradation processes that begin immediately after a sample is removed from the body. These include autolysis, where the tissue’s own enzymes break down cellular components, and putrefaction, caused by microorganisms.
By stopping these degenerative processes, fixation aims to maintain cells and tissue components in a state closely resembling their living form, essentially creating a stable “snapshot” at the cellular level. This allows for accurate observation of cellular morphology and chemical patterns. Furthermore, fixation physically hardens the soft tissue, making it resilient enough to withstand the mechanical stresses of subsequent processing steps, such as cutting into thin slices.
The Fixation Process
A tissue sample’s journey begins the moment it is removed from the body. Speed is important in transferring it to a fixative solution to minimize degradation before preservation begins. The fixative then penetrates the tissue, initiating chemical and physical changes that stabilize its structure.
Several factors influence fixation effectiveness. Tissue size and thickness are important; tissues should be thin enough for adequate penetration. A sufficient volume of fixative is also necessary, typically a minimum of 15 to 20 times the tissue volume, to ensure full immersion.
Temperature can affect the rate of fixative diffusion and chemical reactions; while room temperature is common, elevated temperatures can accelerate the process, though this also carries a potential for increased degradation in unfixed areas. The duration of immersion, commonly ranging from 6 to 24 hours, depends on the tissue size and the specific fixative used, allowing sufficient time for penetration and chemical reactions to reach equilibrium.
Common Fixation Methods
Tissue preservation can be achieved through chemical or physical means, each with distinct applications. Chemical fixation is the most widely used approach, relying on various reagents to stabilize cellular components. Formaldehyde, commonly used as neutral buffered formalin, is the most common chemical fixative in histology laboratories.
Formaldehyde works by forming cross-links between proteins, reacting with amino acid residues to create bridges. This establishes a stable molecular scaffold within the tissue, preventing the dissolution and movement of cellular components. While formaldehyde penetrates tissue quickly, the cross-linking reactions are slower.
Other chemical fixatives include alcohols, such as ethanol, which coagulate proteins; however, prolonged exposure can lead to tissue shrinkage and excessive hardening. Glutaraldehyde, another aldehyde, penetrates more slowly but is more effective at forming extensive cross-links, making it the preferred fixative for preserving ultrastructure in electron microscopy, though it can hinder routine staining methods. Picric acid also precipitates proteins and is often used for connective tissue and glycogen preservation.
Physical fixation methods also exist, with cryofixation, or rapid freezing, being a prominent example. In this technique, tissue is quickly frozen to very low temperatures, often by immersion in a liquid coolant. This method is primarily used when rapid analysis is needed, such as for intraoperative diagnosis during surgery. A significant advantage of cryofixation is its speed, but a disadvantage is the potential for ice crystal formation within the tissue, which can distort cellular morphology.
Next Steps in Tissue Analysis
Once tissue has undergone fixation, it is prepared for microscopic examination through several subsequent stages. The first is embedding, where the fixed tissue is infiltrated with a supportive medium, most commonly paraffin wax, and then encased within a block. This process provides the tissue with the rigidity necessary for precise sectioning.
Following embedding, the tissue block is ready for sectioning, a process performed using a specialized instrument called a microtome. This device precisely slices the wax block into extremely thin, transparent sections. These sections are then mounted onto glass slides.
The final stage before microscopic viewing is staining. Because most cellular components are naturally colorless, dyes are applied to the tissue sections to make them visible and distinguishable under a microscope. Common staining techniques, such as Hematoxylin and Eosin (H&E), use different dyes that bind to specific cellular structures, highlighting various components like nuclei and cytoplasm, completing the tissue’s journey from a living sample to a diagnostic slide.