Fixation, Decalcification & Tissue Processing Essentials

Vocabulary flashcards covering major terms and concepts from fixation through trimming in histopathologic tissue processing.

Fixation

Process that kills, hardens and preserves tissues for microscopic study, preventing degeneration, putrefaction, autolysis and distortion.

Fixative

Chemical agent used in fixation to stabilize cellular structures and constituents.

Coagulation (in Fixation)

Precipitation of tissue and cell components, producing a network that immobilizes proteins.

Preservation (in Fixation)

Maintenance of tissues in a state nearly like the living condition by forming stable chemical compounds.

Effects of Fixatives

Harden tissues, protect from osmotic damage, inhibit bacteria, improve optical contrast, reduce infection risk.

Characteristics of a Good Fixative

Inexpensive, stable, safe, rapid action, inhibits autolysis, minimal shrinkage, penetrates quickly, hardens tissue, isotonic while rendering components insoluble.

Simple Fixative

Fixative composed of a single chemical component.

Compound Fixative

Mixture of two or more fixatives combined to optimize preservation effects.

Microanatomical Fixative

Preserves overall tissue architecture without altering normal intercellular relationships.

Cytological Fixative

Specifically preserves cellular details or particular cell elements.

Histochemical Fixative

Maintains chemical constituents for subsequent histochemical reactions.

Neutral Buffered 10% Formalin

Standard fixative (4% formaldehyde) buffered to neutral pH; rapid penetration, minimal shrinkage, inexpensive.

Physical Fixation

Uses heat, microwaves, or cryopreservation to stabilize tissues.

Chemical Fixation

Most common mode; tissue is immersed in or perfused with a chemical fixative.

Immersion Fixation

Tissue block is simply soaked in fixative solution.

Perfusion Fixation

Fixative is injected through blood vessels to fix whole organs or small animals.

Size & Thickness (Fixation Factor)

Thicker specimens slow penetration; optimal ≤4 mm for light microscopy.

Decalcification

Removal of calcium salts from bone or calcified tissue after fixation to allow sectioning.

Decalcifying Agent

Chemical or physical method used to dissolve or remove mineral content.

Acid Decalcifiers

Strong or weak acids (e.g., formic, nitric) that rapidly dissolve calcium.

Chelating Agent

Substance (e.g., EDTA) that binds calcium ions slowly but gently for decalcification.

Ion Exchange Resin

Resin that removes released calcium ions from solution to speed weak-acid decalcification.

Electrophoresis (Decalcification)

Electric current drives calcium ions out of tissue, accelerating decalcification.

Dehydration

Removal of water from tissue after fixation and before wax infiltration.

Dehydrating Agent

Liquid (commonly alcohol) that replaces tissue water during processing.

Ideal Dehydrating Agent

Rapid, low volatility, penetrates fat, minimal hardening, non-toxic, doesn’t remove dyes.

Ethyl Alcohol

Preferred routine dehydrant; miscible with water, rapid, good penetration.

Methyl Alcohol

Toxic dehydrant mainly for blood smears and cytologic films.

Butyl Alcohol

Slow dehydrant used in plant and animal microtechnique.

Clearing

Replacement of dehydrating agent with a fluid miscible with paraffin, rendering tissue translucent.

Clearing Agent

Solvent (e.g., xylene) that removes alcohol and prepares tissue for infiltration.

Ideal Clearing Agent

Miscible with alcohol & paraffin, minimal shrinkage, slow evaporation, makes tissue transparent.

Xylene

Colorless routine clearing agent; fast (15-60 min) but causes brittleness in large blocks.

Benzene

Rapid clearing solvent with minimal shrinkage but highly toxic and carcinogenic.

Toluene

Xylene substitute producing softer tissue; less carcinogenic but still toxic.

Chloroform

Slow clearing agent good for tough tissues; less brittle but hepatotoxic.

Cedarwood Oil

Gentle, very slow clearing agent ideal for CNS and delicate tissues; no distortion.

Infiltration (Impregnation)

Replacement of clearing agent with a molten medium (usually paraffin) that hardens for support.

Paraffin Wax

Most common infiltration & embedding medium; melts 50–60 °C, inexpensive, easy to section.

Paraffin Wax Advantages

Allows thin sections, rapid (24 h), long-term storage, compatible with many stains.

Paraffin Wax Disadvantages

Overheating brittles tissue; prolonged impregnation causes shrinkage; not ideal for fatty or hard tissues.

Vacuum Infiltration

Impregnation under negative pressure to speed wax penetration and remove bubbles.

Paraplast

Paraffin-plastic polymer blend (mp 56–57 °C) that provides improved sectioning support.

Embedding

Placement of infiltrated tissue into a mold with medium (e.g., paraffin) to solidify in correct orientation.

Leuckhart’s Embedding Mold

Adjustable L-shaped metal strips forming a customizable paraffin mold.

Tissue-Tek System

Embedding unit with heated work plate and −5 °C cold plate for rapid block solidification.

Trimming

Cutting excess wax from a solid block to expose tissue face prior to microtomy.

Plane-Concave Knife

Microtome blade with one flat and one concave side; 250 mm length, for celloidin sections.

Biconcave Knife

Both sides concave; 120 mm blade suited for paraffin sections on rotary microtomes.

Plane-Wedge Knife

Straight-sided 100 mm blade used for frozen sections and hard specimens.