Chemical fixation is a foundational technique in biological and medical sciences, serving as the necessary initial step to prepare tissue and cell samples for microscopic analysis. The process involves treating biological material with chemical agents to stabilize cellular components and prevent decay. This stabilization maintains the sample’s architecture, allowing researchers and pathologists to accurately study the organization of cells and tissues as they existed in the living organism.
Preserving Biological Structures
The goal of chemical fixation is to halt the rapid deterioration that begins the moment a sample is removed from a living system. Unchecked, biological tissues quickly undergo two destructive processes: autolysis and putrefaction. Autolysis, or “self-digestion,” occurs when the cell’s internal enzymes, such as proteases and nucleases, are released and begin to break down cellular components.
These enzymes degrade proteins, lipids, and nucleic acids after cell death, leading to a loss of structural detail. Putrefaction involves the breakdown of the tissue by external bacteria and fungi, which further decompose the organic matter. Together, these processes quickly destroy the sample’s original morphology, leading to structural artifacts and making research unreliable.
Fixation stops these destructive processes by rapidly deactivating the internal enzymes and physically stabilizing the tissue matrix. This stabilization maintains the morphology of the cells and surrounding structures, ensuring the microscopic image accurately reflects the living state. This chemical intervention also increases the mechanical strength of the tissue, which is beneficial for subsequent processing steps, such as cutting thin sections for microscopy.
The Molecular Action of Fixatives
Chemical fixatives achieve preservation through one of two primary molecular mechanisms: cross-linking or denaturation and precipitation. Cross-linking fixatives, notably the aldehydes, chemically bridge molecules together to form a stable, interconnected network. Formaldehyde, for example, reacts with the amino groups on proteins, creating methylene bridges that link adjacent protein chains both within a single molecule and between different molecules.
This cross-linking stabilizes the entire cellular matrix. The extensive chemical bonding stiffens the tissue, inactivates destructive enzymes, and prevents soluble components from dissolving or diffusing away. Glutaraldehyde, a larger molecule with two reactive aldehyde groups, is a more potent cross-linker than formaldehyde and is highly effective for preserving ultra-fine structural details.
The second major mechanism is denaturation and precipitation, commonly employed by organic solvent fixatives like ethanol and methanol. These agents remove water from the tissue, disrupting the tertiary structure of proteins. This process causes the proteins to rapidly unfold and coagulate, or precipitate, into an insoluble mass.
The resulting protein coagulation creates a permeable, mesh-like network that traps other cellular components. While this method is fast and beneficial for specific molecular studies, the rapid dehydration can sometimes lead to tissue shrinkage and distortion of cellular morphology compared to cross-linking fixatives.
Standard Chemical Agents and Applications
The choice of fixative depends on the type of tissue and the specific analytical method planned. Aldehyde-based fixatives are the most common group used in routine laboratories due to their broad utility and excellent preservation of general tissue morphology. Neutral buffered formalin (10% formaldehyde solution) is the standard fixative for histology because it is flexible and compatible with a wide range of subsequent staining and molecular techniques.
For studies requiring extremely high resolution, such as transmission electron microscopy, glutaraldehyde is preferred because its superior cross-linking preserves the finest ultrastructural details of organelles. However, glutaraldehyde’s strong cross-linking can interfere with some staining and molecular analyses, making it unsuitable for routine pathology.
Non-aldehyde fixatives, which operate through denaturation, are used when preserving nucleic acids or specific protein structures is prioritized over overall morphology. Ethanol and methanol are frequently used for cytology smears and molecular studies because they preserve DNA and RNA integrity better than cross-linking agents. These alcohol-based fixatives are often necessary when subsequent analysis, such as immunohistochemistry, requires the antigen’s structure to remain unaltered by chemical cross-links.