Histology Fixation Process

Study Notes

Fixation stabilizes proteins in biological tissue, preventing decay through autolysis (self-digestion) or putrefaction (bacterial decay).
Fixatives are chemicals that halt biochemical reactions and increase tissue strength/stability.
Fixatives prevent postmortem degeneration, including autolysis and putrefaction, and maintain the relationship between cells and extracellular substances (e.g., collagen, elastin).
They enhance tissue visibility by altering refractive indexes. Fixation aims to preserve tissue as close to its natural state as possible for examination.

Fixatives improve tissue receptiveness to dyes, often acting as mordants (linking dyes to tissue).
Different fixatives create unique morphologic patterns and artifacts (visible changes).
Physical fixation methods include heat (e.g., microwave) and desiccation (air drying), though less common in histology.
Chemical fixation is primary, using additive or non-additive fixatives, and coagulant or non-coagulant fixatives.
Additive fixatives chemically bind to tissue; examples include mercuric chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, zinc sulfate, and zinc chloride.
Non-additive fixatives (e.g., acetone, alcohols) dissociate water from proteins, potentially causing shrinkage and hardening.
Coagulant fixatives create a tissue network for easy solution penetration (e.g., zinc salts, mercuric chloride, cupric sulfate, ethyl/methyl alcohol, acetone, picric acid, acetic acid [for nucleic acids]).
Non-coagulant fixatives create gels hindering solution penetration (e.g., formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, potassium dichromate, acetic acid).

Temperature affects tissue morphology.
Tissue thickness is crucial; samples should be no thicker than 3mm for optimal reagent penetration.
The volume ratio of fixative to tissue is important (15-20 times greater fixative volume than tissue volume).
Fixation time is critical; tissue should be placed in fixative immediately after removal, and adequate fixation time (e.g., 6-8 hours for formalin) is necessary for morphology preservation to prevent distortion during subsequent processing.