3. Decalcification, Dehydration, and Clearing

Decalcification

The process of removing calcium or lime salts from hard tissues

20:1

Ratio of decalcifying agent to tissue

1-2 days

General duration for decalcification

Acid Decalcifying Agents

Most widely used as they are stable, easily available, and relatively inexpensive

Acid Decalcifying Agents

Strong mineral acids will cause loss of nuclear staining and macerate tissue if used longer than necessary

Nitric Acid

Strong mineral acid; most common and fastest decalcifying agent; can be used with other reagents or simply as an aqueous solution with 5-10% concentration

10% Aqueous Nitric Acid

Produces good nuclear staining and minimal distortion; easily removed by 70% alcohol; recommended for urgent biopsies and imparts a yellow color with nitrous acid

12-24 hrs

Duration of decalcification using 10% aqueous nitric acid solution

Formol-Nitric Acid

Produces less tissue destruction than 10% aqueous nitric acid; should be used inside a fume hood and imparts a yellow color with nitrous acid

1-3 days

Duration of decalcification using formol-nitric acid

Perenyi’s Fluid

Decalcifies and softens tissue recommended for routine purposes; not recommended for urgent biopsies; nuclear and cytoplasmic staining is good2

2-7 days

Duration of decalcification using Perenyi’s Fluid

Phloroglucin-Nitric Acid

Most rapid decalcifying agent; recommended for urgent works but causes poor nuclear staining

12-24 hrs

Duration of decalcification using phloroglucin-nitric acid

Hydrochloric Acid

Inferior to nitric acid due to its slower action and greater distortion of tisse

Hydrochloric Acid

Produces good nuclear staining at 1% concentration with 70% alcohol; good for surface decalcification of tissue blocks

Von Ebner’s Fluid

Moderately rapid decalcifying agent recommended for teeth and small pieces of bone

Von Ebner’s Fluid

Permits relatively good cytologic staining and does not require washing out before dehydration

Formic Acid

Weak acid; produces better nuclear staining and safer to handle; recommended for post-mortem research tissues, small pieces of bone and teeth

Formic Acid

Decalcifying agent suitable for immunohistochemical staining; both a fixative and decalcifying agent

Formic Acid

Best decalcifying agent

Formic Acid

Moderate-acting but slow decalcifying agent; addition of citrate hastens decalcification; gentle in action and less likely to interfere with nuclear staining

2-7 days

Duration of decalcification using formic acid

Formic Acid-Sodium Citrate Solution

Relatively slow decalcifying agent which permits better nuclear staining than nitric acid; recommended for autopsy materials, bone marrow, cartilage, and tissue for research purposes

3-14 days

Duration of decalcification using formic acid-sodium citrate solution

Trichloroacetic Acid

Weak acid and a slow decalcifying agent which is both a fixative and decalcifying agent and permits good nuclear staining; suitable for small spicules of bones

4-8 days

Duration of decalcification using trichloroacetic acid

Sulfurous Acid

Very weak decalcifying agent suitable for minute pieces of bone

Chromic Acid (Flemming’s Fluid)

Both a fixative and decalcifying agent which can be used for minute bone spicules but carcinogenic and highly corrosive to skin and mucous membrane

Citrate Acid-Citrate Buffer Solution

Decalcifying agent that is too slow for routine purposes and permits excellent nuclear and cytoplasming staining; does not produces cell or tissue distortion

6 days

Duration of decalcification using citric acid-citrate buffer solution

Chelating Agents

Substances that combine with calcium ions and other salts to form weakly dissociated complexes and facilitate the removal of calcium salts

EDTA

Most common chelating agent

1-3 weeks

Duration of chelation for small specimens

6-8 weeks

Duration of chelation for dense bones

Ion Exchange Resin

Ammonium form of polystyrene resin

Ion Exchange Resin

Hastens decalcification by removing calcium ion from formic-containing decalcifying agents which increases solubility

Ion Exchange Resin

Not recommended for fluids containing mineral acids such as nitric and hydrochloric acid

Electrophoresis

Positively-charged calcium ions become attracted to negative electrodes, which removes them from the decalcifying solution

88% Formic Acid

Used for electrophoresis

Electrophoresis

Suitable for small bone fragments

• Concentration of the decalcifying agent

• Fluid access to the tissue

• Size and consistency of the tissue

• Agitation

• Temperature

Factors that influence the rate of decalcification

Physical or Mechanical Method

Done by touching or bending of the tissue with the fingers to determine its consistency or by pricking the tissue with a fine needle or probe

Chemical Method

Simple, reliable and convenient method that is recommended for routine purposes

Chemical Method

Involves the detection of calcium in acid solutions through precipitation of insoluble calcium oxalate

Incomplete Decalcification

Presence of cloudiness

Complete Decalcification

Clear solution

X-Ray or Radiological Method

Very expensive but most ideal, most sensitive, and most reliable method

X-Ray or Radiological Method

Not recommended for mercuric chloride-fixed tissues due to the occurrence of radio-opacity which can interfere with correct interpretation

• Immersing the decalcified tissues to saturated lithium carbonate or 5-10% aqueous sodium bicarbonate

• Rinsing the decalcified tissues in running tap water

• For frozen sections, wash with water or store in formol-saline with 15% sucrose or phosphate-buffered saline with 15-20% sucrose at 4 deg C before freezing

Post-Decalcification methods:

Surface Decalcification

For small unexpected deposits of calcium encountered in paraffin blocks

Perenyi’s fluid

May act as decalcifying agent and tissue softener

Lendrum’s fluid

4% aqueous phenol solution

Molliflex

Tissue softener which makes tissues appear swollen and soapy

Dehydration

The process of removing intracellular and extracellular water from the tissue following fixation and prior to infiltration

10:1

Ratio of dehydrating agent to tissue

Alcohol

Most common dehydrating agent

Ethyl Alcohol

Best dehydrating agent; clear, colorless, flammable; fast-acting, able to mix with water and many organic solvents, and penetrates tissue easily

Ethyl Alcohol

Recommended for routine tissue dehydration

Methyl Alcohol

Dehydrating agent primarily for blood and tissue films, and smear preparations but is toxic and can cause blindness and death

Butyl Alcohol

Dehydrating agent used in plant and animal microtechniques; slow but produces less shrinkage; odorous

Tertiary Butanol

Known as a universal solvent; acts as both a dehydrating and clearing agent

Tertiary Butanol

Dehydrating agent that tends to solidify at room temperature or below 25 deg C

Amyl Alcohol

Dehydrating agent that is miscible with 90% alcohol, toluene, and xylene; dissolves paraffin

Isopropyl Alcohol

Dehydrating agent that is an excellent substitute for ethanol

Isopropyl Alcohol

Dehydrating agent not used for preparing staining solutions; best clearing agent for microwave techniques

Acetone

Clear, colorless, cheap and rapid acting; Dehydrating agent utilized for most urgent biopsies

Acetone

Dehydrating agent with poor penetration and causes brittleness and shrinkage; Highly flammable and volatile; Lipids are removed from the tissue but only for small pieces of tissue; not recommended for routine processes

30 mins to 2 hrs

Duration of dehydration using acetone

Dioxane (Diethylene Dioxide)

Excellent dehydrating and clearing agent with less tissue shrinkage than alcohol

Dioxane (Diethylene Dioxide)

Miscible with water, paraffin, alcohol and xylol; Ribbon poorly, extremely dangerous and expensive; Tissues can be left for longer periods of time

Graupner’s Method

Makes use of pure dioxane and paraffin wax; Uses a specific time schedule for dehydration

Weiseberger’s Method

Tissue is wrapped in a gauze bag and suspended in a bottle containing dioxane and anhydrous calcium oxide

3-24 hrs

Duration of dehydration using Weiseberger’s method

Cellosolve (Ethylene glycol monoethyl ether)

Dehydrates rapidly and can store tissue for months without producing hardening or distortion

Triethyl Phosphate

Rapid dehydrating agent and produces very little distortion, shrinkage and hardening; soluble in alcohol, water, ether, benzene, chloroform, acetone and xylene

Triethyl Phosphate

Used to dehydrate sections and smears following certain stains

Tetrahydrofuran

Dehydrates and clears at the same time; miscible in both water and paraffin; Dissolves many substances including fats but does not dissolve out aniline dyes which results to improved staining

Universal Solvent

Solutions that can act as both a dehydrating and clearing agent

• Tertiary butanol

• Dioxane

• Tetrahydrofuran

Examples of universal solvent

Clearing

Transition step between dehydration and infiltration

Clearing

The process of removing the dehydrating agent from the tissues and replacing it with an agent that should be miscible to both alcohol and paraffin

Hydrocarbons

Odorless flammable liquids with characteristic petroleum or aromatic odors

Esters

Colorless flammable solvents miscible with most organic solvents

Terpenes

Isoprene polymers found in essential oils origannaly derived from plants and are the earliest transition solvents to be used

Xylene

Clear rapidly and render tissues transparent; most rapid clearing agent

30 mins to 1 hr

Duration of clearing using xylene

Xylene

Evaporates quickly in paraffin oven which makes impregnation and embedding faster; becomes milking when incompletely dehydrated tissue is immersed in it; reasonably cost-effective and works well for short-term clearing of small tissue blocks

Toluene

Does not make tissues excessively hard and brittle even if left for 24 hyrs

Toluene

More tolerant of small amounts of water left on the tissue; more expensive and more toxic than xylene, but not carnogenic.

1-2 hrs

Duration of clearing using toluene

Benzene

Penetrate and clears tissues rapidly; a popular routine clearing egent until it was found to be highly carcinogenic; does not make tissues hard and brittle and causes minimum shrinkage

Chloroform

Slower in xtain than xylene but causes less brittleness; tissues do not become translucent; recommended for tough tissues, nervous tissues, lymph nodes and embryos; suitable for large tissue specimens

6-24 hrs

Duration of clearing using chloroform

Cedarwood Oil

Able to clear both paraffin and celoidin sections; recommended for CNS tissues and cytological studies such as smooth muscles and skin

Cedarwood Oil

Tissue may be left in old indefinitely without considerable damage and distortion; does not interfere too seriously with paraffin penetration if not completely removed

2-3 days

Duration of clearing using cedarwood oil

Aniline Oil

Recommended for clearing embryos, insects, and very delicate specimens due to its ability to clear 70% alcohol

Clove Oil

Causes minimum shrinkage but quality is not guaranteed due to its tendency to become adulterated

Clove Oil

Wax impregnation after clearing is slow and difficult; unsuitable for routine clearing purposes