Fixation and Fixative Agents

Vocabulary flashcards covering key terms, agents, types, factors, and specific formulations involved in tissue fixation for histology and cytology.

Fixation

Process that stabilizes cellular and tissue constituents in a physical and chemical state to withstand subsequent processing with minimal loss or distortion.

Fixatives

Fluids, often chemical mixtures, that denature and cross-link proteins to prevent autolysis, harden tissue, and preserve morphology.

Volume (Fixation Factor)

Fixative should be 10–20 times tissue volume (50–100:1 for museum specimens); agitation speeds penetration.

pH / Hydrogen Ion Concentration

Optimal range 6–8; acidity encourages formalin-heme pigment formation in blood-rich tissue.

Temperature (Fixation Factor)

40 °C for routine processing; 0–4 °C for EM; room temperature adequate; 60 °C formalin for urgent biopsies.

Section Thickness

LM ≤ 4 mm thick; EM 1–2 mm²; solid organs ≤ 10–15 mm long; whole brain fixed intact in formalin 2–3 weeks.

Osmolality

Hypertonic solutions shrink cells; hypotonic swell and lyse; best fixation at 400–450 mOsm (slightly hypertonic).

Fixative Concentration

Use the lowest effective concentration; excessive strength can mimic heat artefact and damage tissue.

Warm Ischemia Time

Period of anoxic stress after blood supply interruption but before tissue removal.

Cold Ischemia Time

Interval between tissue removal and fixation during which oxygen is absent but metabolism continues.

Fixation Time

Duration tissue remains in fixative; influences penetration and cross-linking quality.

Effects of Fixatives

Reduce infection risk, harden tissue, prevent autolysis, inhibit bacteria, enhance staining, and may act as mordants.

Characteristics of a Good Fixative

Cheap, stable, safe, rapid-acting, inhibits decomposition, minimizes shrinkage, penetrates evenly, hardens, and allows diverse staining.

Aldehyde Fixatives

Group including formaldehyde and glutaraldehyde; act by cross-linking proteins.

Oxidizing Agents (Fixatives)

Fixatives such as osmium tetroxide and potassium permanganate that oxidize tissue components.

Alcohol-Based Fixatives

Fixatives like methyl or ethyl alcohol and acetic acid that precipitate proteins.

Metallic Fixatives

Solutions containing metals (e.g., mercuric chloride, picric acid) that aid preservation and staining.

Simple Fixatives

Single-component solutions such as formaldehyde, acetone, acetic acid, or osmium tetroxide.

Compound Fixatives

Mixtures of two or more fixatives blended for combined optimal effects.

Microanatomical Fixatives

Fixatives aimed at preserving overall tissue architecture, e.g., 10 % NBF, Bouin’s, Zenker’s.

Cytological Fixatives

Fixatives tailored for cellular detail; subdivided into nuclear, cytoplasmic, and histochemical types.

Cross-Linking Fixatives

Agents that form covalent bonds between proteins, stabilizing tissue (e.g., aldehydes).

Precipitating / Denaturing Fixatives

Agents that lower protein solubility and disrupt hydrophobic interactions, causing coagulation.

Additive Fixatives

Chemicals that chemically combine with tissue (e.g., formaldehyde, picric acid, zinc sulfate).

Non-Additive Fixatives

Do not form chemical bonds with tissue; examples include acetic acid, acetone, and alcohols.

Coagulant Fixatives

Cause protein precipitation forming a meshwork (e.g., mercuric chloride, picric acid, zinc salts).

Non-Coagulant Fixatives

Stabilize proteins without visible precipitation (e.g., formaldehyde, glutaraldehyde).

Formaldehyde (Formalin)

37–40 % gas solution; used as 10 % neutral-buffered formalin (4 % formaldehyde) for routine fixation.

Advantages of Formalin

Cheap, penetrates well, compatible with stains, preserves fat, mucin, glycogen, and avoids brittleness.

Disadvantages of Formalin

Irritating, tissue shrinkage, pigment formation in unbuffered form, prolonged fixation can bleach tissue and dissolve glycogen.

10 % Formal Saline

Buffered formalin (pH 6.8); 12–24 h fixation; good for CNS, post-mortem tissue, and lipid preservation.

10 % Neutral-Buffered Formalin (NBF)

Phosphate-buffered formalin (pH 7.4); 4–24 h fixation; ideal for routine surgical specimens and iron pigments.

Zinc Formalin

Unbuffered zinc sulfate plus formalin; mercury-free alternative suitable for immunohistochemistry.

Formol-Corrosive (Formol-Sublimate)

Mixture of saturated mercuric chloride and formalin; used for routine autopsy tissue (≤ 1 cm sections).

Paraformaldehyde

Polymerized formaldehyde; dissolves in water to yield monomer; excellent for antigen preservation and avoids pigment.

Karnovsky’s Fixative

Buffered mixture of paraformaldehyde and glutaraldehyde; used for resin embedding in LM and EM.

Glutaraldehyde

Dialdehyde fixative; 2.5 % for small biopsies, 4 % for larger pieces; gold standard for electron microscopy.

Factors Affecting Formalin Fixation

Include post-mortem interval, fixative composition, volume, time, temperature, tissue thickness, and storage conditions.