Tissue fixation is a standard procedure in biology and medicine used to preserve biological samples for later study. Without this process, delicate cellular structures would rapidly degrade, making accurate analysis impossible. The most common fixative employed globally is formalin, a dilute, aqueous solution of the chemical formaldehyde. This approach chemically stabilizes tissues, allowing researchers and pathologists to examine cell morphology and molecular components under a microscope. The goal of fixation is to maintain the tissue’s structure as closely as possible to its living state.
Why Tissue Preservation Is Necessary
Immediately after tissue is removed from a living organism, a rapid process of degradation begins. This initial breakdown is driven by autolysis, a form of “self-digestion” where the tissue’s own intracellular enzymes, particularly lysosomal hydrolases, are released and begin to break down proteins and nucleic acids. If this enzymatic activity is not stopped quickly, the entire cellular architecture collapses, distorting the true appearance of the cells.
Simultaneously, microorganisms present in or on the tissue, such as bacteria, initiate a process called putrefaction. These microbes metabolize the organic material, further accelerating the decomposition. Fixation is necessary to halt both autolysis and putrefaction instantly, effectively freezing the tissue in a life-like state. By stabilizing the cellular components, fixation ensures that subsequent preparation, sectioning, and staining steps accurately reflect the tissue’s structure at the time of removal.
The Chemical Reaction That Stabilizes Cells
Formaldehyde, the active component in formalin, is a small, highly reactive organic molecule that acts primarily by stabilizing proteins. Its chemical structure includes a single carbon atom double-bonded to an oxygen atom, making it an aldehyde. This small size allows the molecule to rapidly penetrate cell membranes and diffuse throughout the entire tissue sample.
Once inside the cell, the formaldehyde molecule initiates cross-linking, which is the defining mechanism of fixation. Formaldehyde reacts specifically with the amino groups (NH2) found in the side chains of proteins, particularly the amino acid lysine. This initial reaction forms an intermediate compound called a hydroxymethyl group (-CH2OH) attached to the protein.
The hydroxymethyl intermediate is highly reactive. This intermediate then reacts with another adjacent amino group on a different protein molecule, or sometimes with other groups like amides or sulfhydryls. This secondary reaction results in the formation of a stable methylene bridge (-CH2-) that physically links two separate protein molecules together.
These newly formed chemical bonds lock the proteins into a rigid, three-dimensional mesh, effectively stiffening the entire cellular and extracellular matrix. The extensive cross-linking makes the proteins insoluble in water, preventing them from dissolving or changing shape during subsequent tissue processing steps. Furthermore, by forming these cross-links, formaldehyde alters the tertiary structure of enzymes, rendering them inactive. This inactivation stops the self-digestion of autolysis, as the destructive hydrolase enzymes can no longer function. The overall effect is a preserved specimen where cellular organelles and architectural relationships are maintained.
Preparing and Handling Fixed Tissue
Achieving optimal tissue preservation requires attention to several practical details during the preparation phase. The standard clinical and research formulation is 10% Neutral Buffered Formalin (NBF), which contains about 4% formaldehyde. This concentration provides a good balance between fixation speed and minimizing tissue hardening.
Neutral Buffering and pH
The “buffered” aspect is important because the solution must maintain a neutral pH, typically around 7.0. If the solution becomes too acidic, a dark brown pigment called formalin-heme pigment can form in the tissue. This pigment obscures cellular detail and complicates subsequent analysis. Maintaining a neutral pH prevents this unwanted artifact from developing.
Volume Ratio and Penetration
A proper volume ratio between the fixative and the tissue is also important. Ideally, the volume of the 10% NBF should be at least 10 to 20 times greater than the volume of the specimen. This large ratio ensures that the formaldehyde is not rapidly diluted by the water content of the tissue, maintaining an effective concentration.
Duration and Safety
The duration of fixation must be carefully monitored, as it depends on the size and density of the specimen. For small biopsies, fixation may be complete within 6 to 8 hours, but larger surgical specimens may require 24 to 48 hours or even longer to ensure complete penetration and stabilization. Incomplete fixation can lead to artifacts where the center of the tissue appears distorted because the interior cells began to autolyze before the fixative reached them. Given that formaldehyde is a known toxic and hazardous substance, proper ventilation and personal protective equipment are standard laboratory requirements when handling the fixative and fixed tissues.