Tissue fixation is the first step in preparing a biological sample for microscopic analysis, a process known as histology. This procedure aims to chemically or physically stabilize the tissue, preserving its structure and molecular components close to their living state. By halting biological activity, fixation ensures that the intricate cellular architecture, or morphology, remains intact for study and diagnosis. Preservation of this architecture is fundamental because all subsequent steps, from processing to staining, rely on the tissue’s initial stability.
Why Immediate Fixation is Critical
Once tissue is removed from the body, it immediately begins self-destruction driven by two simultaneous processes: autolysis and putrefaction. Autolysis is the “self-digestion” of cells, initiated when lack of blood supply causes oxygen deprivation and a drop in cellular pH. This leads to the release of hydrolytic enzymes from within the cells’ lysosomes.
These enzymes break down cellular components, rapidly destroying the fine structural details needed for accurate examination. Putrefaction involves decomposition caused by external agents like bacteria and fungi. Immediate fixation terminates the activity of these destructive enzymes and kills microorganisms, freezing the tissue’s structure before it degrades into an unusable state.
Chemical Versus Physical Stabilization
Fixation is achieved through two primary approaches, each suited for different analytical goals. Chemical fixation, the most common method, involves immersing the tissue in a solution that chemically alters its components. Ten percent neutral buffered formalin is the standard in histology.
This chemical method is favored for preserving overall tissue morphology and for long-term storage, as the changes create a durable, stable sample. Physical fixation typically involves cryofixation, or snap-freezing the tissue, often in liquid nitrogen. This technique is preferred when the goal is to preserve cellular components in their native state for analyses like immunohistochemistry or enzyme activity studies.
While freezing provides a rapid stop to biological processes, it often results in structural artifacts, such as ice crystal damage, compared to chemical fixation.
How Fixatives Change Tissue Structure
Chemical fixatives stabilize tissue by reacting with macromolecules, primarily proteins, to create a stable, insoluble network. Aldehyde-based fixatives like formalin form cross-links, which are covalent bridges between adjacent protein molecules. This cross-linking transforms the soft cellular material into a rigid gel that resists distortion during subsequent processing steps.
Formaldehyde penetrates the tissue quickly and binds to amino acids, slowly forming methylene bridges between protein chains. This chemical hardening provides the mechanical strength necessary to withstand the harsh solvents used later in preparation. Physical fixation achieves stability by rapidly lowering the tissue temperature below the freezing point of water, halting molecular movement and enzymatic reactions without chemical modification.
From Fixed Sample to Microscope Slide
The fixed tissue must undergo conditioning steps before it can be thinly sliced for microscopic viewing. The first step is dehydration, where water and residual fixative are gradually removed by immersing the tissue in increasing concentrations of alcohol. This is necessary because the subsequent embedding medium, typically paraffin wax, is hydrophobic and immiscible with water.
Following dehydration, the tissue moves to the clearing step, where an organic solvent like xylene removes the alcohol. The tissue becomes translucent, making it receptive to the final embedding medium. The final stage is infiltration and embedding, where the cleared tissue is placed into molten paraffin wax. The wax penetrates the tissue structure and solidifies to form a supportive block. This solid block provides the rigid matrix needed to hold the tissue intact while it is sliced into sections, often as thin as 5 micrometers, using a microtome, before being placed on a glass slide for staining.