Fixation in histopathology. Learn the purpose, mechanisms, types of fixatives (like formalin), factors affecting fixation, and procedures. Essential reading for medical and histology students. Includes FAQ for common troubleshooting.

What is Fixation ?

When tissue is removed from the body, it undergoes post-mortem changes, which can be putrefactive (caused by bacterial invasion) or autolytic (due to enzymes from dead cells). Fixation is a process that safeguards tissue from these changes by lowering temperature or using chemicals. Key points include:

• Putrefaction involves bacteria from the alimentary tract causing decomposition.
• Autolysis mainly occurs in the CNS and endocrine system due to enzyme action.

Aims and Purpose of Fixation

The objectives of effective fixation are multifactorial:

1. Prevention of Decomposition: To halt the activity of degradative enzymes and putrefactive bacteria.
2. Hardening and Stabilization: To solidify the tissue, making it firm enough to withstand subsequent processing and sectioning on a microtome.
3. Preservation of Structure: To maintain the natural architecture of cells and extracellular components (organs, nuclei, cytoplasm) indefinitely.
4. Coagulation of Proteins: To render proteins insoluble, which locks cellular components in place and prevents their diffusion or loss.
5. Optical Differentiation: To alter the refractive index of tissue components, enhancing contrast for microscopic viewing.
6. Enhancing Affinity for Dyes: To modify the tissue chemically so it reacts appropriately with stains (e.g., becoming acidophilic or basophilic).

Mechanisms of Fixation

Fixatives act through several biochemical mechanisms:

• Cross-linking: Fixatives like formaldehyde and glutaraldehyde create covalent methylene bridges (-CH₂-) between protein molecules (primarily between lysine residues). This forms a stable, insoluble gel that preserves structure excellently but can mask some antigens (epitopes).
• Coagulation/Precipitation: Alcohol-based fixatives (e.g., ethanol, methanol) dehydrate the tissue and disrupt hydrogen bonds, causing proteins to denature and precipitate out of solution. This preserves nucleic acids well but can cause significant shrinkage and distortion.
• Oxidation: Fixatives like Osmium tetroxide and Potassium dichromate act as strong oxidizing agents, forming cross-links with proteins and lipids, and are essential for stabilizing phospholipid membranes in electron microscopy.
• Mercuric Chloride Addition: Forms complexes with proteins, leading to excellent nuclear detail but introduces mercury pigments that must be removed (dezenkerization) later.