Decalcification and Tissue Preparation Techniques

Decalcification

is the process of removing calcium deposits (like calcium and lime salts) from tissues, usually bones or teeth, to allow better tissue sectioning for microscopic examination.

Nitric Acid

The most commonly used and fastest decalcifying agent, combined with formaldehyde or alcohol.

Perenyl's Fluid

Acts as both a tissue softener and a decalcifying agent.

Decalcifying Agents

used to remove calcium and lime salts following fixation.

Phloroglucin-Nitric Acid

The fastest decalcifying agent.

Formic Acid

Both a fixative and decalcifying agent, particularly effective for small pieces of bones and teeth.

5% formic acid

It's considered the best general decalcifying agent.

Hydrochloric Acid

Used for surface decalcification of tissue blocks.

Von Ebner's Fluid

is recommended for decalcifying teeth and small bone pieces.

Trichloroacetic Acid

Suitable for small bone spicules. Tissues decalcified with this acid don't need to be washed out, which makes it convenient for small samples.

Sulfurous Acid

A very weak decalcifying agent, primarily used for minute bone pieces.

Chromic Acid

Acts as both a fixative and a decalcifying agent, typically used for small bone spicules.

EDTA (Ethylenediaminetetraacetic Acid)

Removes calcium by binding ions without damaging tissue.

Ion exchange resins

are materials used to remove calcium ions during decalcification while preserving the cellular detail of the tissue.

Physical/Mechanical Test

This method involves bending or piercing the tissue to check for softness, but it is inaccurate and unreliable.

X-ray or Radiological Method

The most ideal, sensitive, and reliable method, but it is expensive. X-rays can detect even small traces of calcium, providing precise results.

Chemical Method

This method is simple, reliable, and recommended for routine use. It involves testing the decalcification solution for the presence of calcium ions. If calcium is still present, the decalcification process isn't complete.

Dehydration

removes both the fixative and water from tissues and replaces them with a dehydrating fluid to prepare them for impregnation.

10:1

The recommended ratio of fluid to tissue volume for dehydration.

Tissue Softeners

for unduly hard tissues THAT may damage the microtome knives.

Alcohol

most common dehydrate.

Ethanol

The most common dehydrating agent, used for routine tissue dehydration.

Methyl Alcohol

Used for blood and tissue films.

Butyl Alcohol

Used in plant and animal microtechniques.

Isopropyl Alcohol

A backup option when ethanol is unavailable.

Acetone

Acts as both a fixative and a dehydrating agent. It's rapid acting, making it useful for urgent biopsies.

Dioxane

Acts as both a dehydrating and clearing agent, and is expensive.

Cellosolve (Ethylene Glycol Monoethyl Ether)

Used for both dehydration and clearing and is toxic and combustible.

Tetrahydrofuran (THF)

A dehydrating and clearing agent that can also dissolve fats.

Triethyl Phosphate

Soluble in various substances (alcohol, chloroform, water, etc.) and used as a dehydrating agent and is universal solvent.

Clearing

is the step in tissue preparation where the dehydrating agent (usually alcohol) is removed from the tissue and replaced with a substance that is miscible with both alcohol and the embedding medium (usually paraffin).

Xylene

Colorless and the most commonly used clearing agent. It is miscible with both alcohol and paraffin. The most rapid clearing agent.

Chloroform

Often used during the embedding process, suitable for thicker tissue block. It does not make the tissue as translucent as xylene.

Benzene

Recommended for urgent biopsies due to its rapid action, miscible with alcohol, does not make tissue brittle, and causes minimal shrinkage, and is carcinogenic.

Toluene

Often used as a substitute for xylene or benzene. Suitable for both embedding and mounting.

Clove Oil

Causes minimal shrinkage of the tissue.

Aniline Oil

Recommended for delicate tissues such as embryos and insects.

Cedarwood Oil

Used for both paraffin and celloidin sectioning. Recommended for CNS (central nervous system) tissues because of its gentle action.

Methyl Benzoate and Methyl Salicylate

Slow-acting clearing agents, often required for double embedding techniques due to their slow clearing process.

Carbon Tetrachloride

Less commonly used today due to its toxicity and potential health hazards, but it was historically used for clearing tissues in some labs.