Dehydration Techniques and Agents in Histology

Dehydration

Process of removing intracellular and extracellular water from tissue.

Ideal Dehydrating Solution

Should dehydrate rapidly without producing considerable shrinkage and distortion of tissues, not evaporate very fast, be able to dehydrate even fatty tissues, not harden tissues excessively, not remove stains, not be toxic to the body, and not be a fire hazard.

Commonly Used Dehydrating Agents

Includes ethanol, acetone, dioxane, cellosolve, triethyl phosphate, and tetrahydrofuran.

Alcohol 30%

Used for dehydration for 1-6 hours.

Alcohol 50%

Used for dehydration for 1-6 hours.

Alcohol 70%

Used for dehydration for 3-12 hours.

Alcohol 90%

Used for dehydration for 3-12 hours.

Absolute alcohol (1)

Used for dehydration for 1-2 hours.

Absolute alcohol (3)

Used for dehydration for 1-2 hours.

Concentrated alcohols

Harden only the surface of tissues.

General rule for dehydrating agent

The amount of dehydrating agent in each stage should NOT be LESS THAN 10 TIMES THE VOLUME of the TISSUE.

Drying

Removal of water by evaporation from a solid, semi-solid or liquid.

Clearing

Also known as dealcoholization; removal of excess alcohol.

Clearing agent

Must be fully miscible with both ethanol and paraffin wax.

Most common clearing agent

Xylene.

Typical Dehydration Sequence for <4mm tissues

1. 70% ethanol - 15 min, 2. 90% ethanol - 15 min, 3. 100% ethanol - 15 min, 4. 100% ethanol - 15 min, 5. 100% ethanol - 30 min, 6. 100% ethanol - 45 min.

Shrinkage

Considerable shrinkage and hardening leading to distortion occurs if formalin-fixed tissues are transferred directly to higher grades of alcohol.

Maceration

Prolonged storage in lower concentrations of alcohol can lead to maceration.

Ethanol

Most commonly used dehydrating agent.

Alcohol 100%

Used for dehydration for 15 min to 45 min in a typical sequence.

Solid tissues

Should NEVER be allowed to air dry.

Minimizing shrinkage

Minimize shrinkage and extraction of cell components by using dehydrating agents in graded series for short periods of time.

Delicate tissues

The more delicate the tissue, the more gently dehydration should be done.

Strength of initial alcohol

Should depend on agents such as acetone, dioxane, cellosolve, triethyl phosphate, and tetrahydrofuran.

37C

Increases dehydration time (for STAT)

BLUE discoloration of copper sulfate crystals

Full saturation of dehydrating fluid with water

Universal solvent

Acts as dehydrating and clearing agent, used in staining series as dehydrating agent, mixes with water, ethanol, xylene, and paraffin

ETHANOL (ETHYL ALCOHOL)

Boiling point: 78.3°C

Ethanol disadvantages

Recommended for routine tissue dehydration, clear, colorless & flammable, doesn't mix to wax, 30% is recommended as starting alcohol in graded alcohol dehydration for delicate tissues, odorous, more expensive than butanol, primary infiltration must be done in half tertiary butanol and half paraffin, prior to paraffin impregnation, solidifies at room temp or below 25°C.

ISOPROPANOL

Boiling point: 82.3°C

Isopropanol advantages

Non toxic, miscible, little shrinkage (use graded alcohols), can be used on eyes & embryo (use graded alcohols), fast acting, best dehydrating solution, reliable, causes less extraction of cellular components, inexpensive & easily obtained, excellent substitute for ethanol, less shrinkage and hardening than ethanol, no government restrictions on its use, water-free to use in place of absolute ethanol, Lillie considers it 'the best all-around substitute for ethyl alcohol', less expensive than tax-free alcohol.

Isopropanol disadvantages

Cannot be used in the celloidin technic since nitrocellulose is insoluble in it, cannot be used for preparing staining solutions, since dyes are not soluble in it, expensive and maybe difficult to obtain, causes excessive shrinkage and hardening in long periods, may have prohibitive taxes, extracts methylene blue & other thiazine dyes, extracts more lipids than acetone, may react with remaining unreduced OsO4, only slightly miscible with most resins.

PENTANOL (AMYL ALCOHOL)

Boiling point: 128°C

BUTANOL (BUTYL ALCOHOL)

Boiling point: 117.7°C

Butanol advantages

Miscible with 90% alcohol, toluene and xylene, dissolves in paraffin wax, utilized in plant & animals' micro-techniques, recommended for tissue that do not require rapid processing.

Butanol disadvantages

Toxic, cannot be used in poorly ventilated rooms, not miscible with water.

ACETONE

Odorous, slow-acting, necessary long period of infiltration, low dehydrating power.

TERTIARY BUTANOL

Boiling point: 82.8°C, boiling point 56°C, cheap rapid acting dehydrating agent, utilized for most urgent biopsies, dehydrates in 1/2 to 2 hours, clear, colorless fluid that mixes with water, ethanol and most organic solvents.

Weiseberger's method

Most lipids are removed from tissues with this dehydrating agent, use is limited because of its volatility and inflammability, not recommended for routine dehydration process.

Weiseberger's method advantages

The tissue is wrapped in a gauze bag and suspended in a bottle containing dioxane and a little anhydrous calcium oxide, water is displaced from the tissue by dioxane and in turn absorbed by calcium oxide or quicklime, dehydration period ranges from 3-24 hours.

Weiseberger's method advantages continued

Rapid dehydrating agent, less expensive than ethanol, does not extract methylene blue and other dyes from stained sections, may cause less shrinkage of specimen than ethanol, not reactive with OsO4 remaining in specimen, miscible with most embedding resins.

Dioxane treatment

Tissues which have been treated with a chromate fixative, e.g. Regaud's or Moller's fluid, should be thoroughly washed in running tap water prior to treatment with dioxane in order to remove chromate.

Disadvantages of Universal Solvent

Requires a clearing agent, volume must be 20 times that of the tissue, best processing requires a graded series of a mixture of acetone and xylene before one can go into paraffin, needs good ventilation, evaporates rapidly, flammable, absolute acetone is easily contaminated with water, resulting in complete dehydration, uranyl acetate and phosphotungstic acid are only soluble in dilute solutions of acetone.

Dioxane

Needs large volume for dehydration, costs about four times more than absolute alcohol, must be used in well-ventilated rooms, cumulatively toxic, odorous, and distorts tissue-containing cavities.

Cellosolve

Refractive index 1.42, boiling point 101.5 °C, excellent dehydrating and clearing agent, readily miscible in water, melted paraffin, alcohol, and xylol, produces less tissue shrinkage compared to alcohol dehydration.

Cellosolve Advantages

Tissues may be placed directly into the solution after washing out, tissue sections dehydrated with dioxane tend to ribbon poorly, extremely dangerous (its vapor produces a cumulative and highly toxic action in man), should not be used routinely, should not be recycled.

Cellosolve Boiling Point

Boiling point 156.4 °C, tissue may be transferred from water or normal saline directly to cellosolve and stored in it for months without producing hardening or distortion.

Cellosolve Toxicity

Combustible at 110-120 °F, toxic by inhalation, skin contact, and ingestion, reproductive, fetal, urinary, and blood systems are particularly vulnerable to their toxic side effects, propylene-based glycol should be used instead of ethylene-based glycol ethers.

Dioxane Graupner's Method

1st Pure Dioxane for 1 hour, 2nd Pure Dioxane for 1 hour, 3rd Pure Dioxane for 2 hours.

Dioxane Advantages

Rapid dehydrating agent, tissue may remain in it for months without injury, avoid distortion and does not require graded dilutions.

Dioxane Disadvantages

Expensive, rapidly absorbs water from the air, requires clearing agent.

Paraffin Wax Processing

1st Paraffin wax for 15 minutes, 2nd Paraffin wax for 45 minutes, 3rd Paraffin wax for 2 hours, embedded in mold and cool in water.

Triethylphosphate

Boiling point 215 °C, removes water very readily and produces very little distortion and hardening of tissue, used to dehydrate sections and smears following certain stains and produces minimum shrinkage.

Propylene Oxide

May be used in routine paraffin technique, displaces water readily with slight distortion, does not harden tissue excessively, may be used as a dehydrating solution in the staining sequence, soluble in alcohol, benzene, toluene, xylene, ether, and chloroform.

Propylene Oxide Disadvantages

None.

Tetrahydrofuran

Transition fluid, completely miscible with embedding resins, can infiltrate tissues readily and reduce the viscosity of embedding tissue resins.

Tetrahydrofuran Hazards

Highly flammable, volatile, toxic, and potentially carcinogenic, very reactive, may cause cytochemical and staining reactions, traces may be retained in polymerized resin.

Acetonitrile

Good substitute for propylene oxide, non-carcinogenic, does not interfere with epoxy polymerization, freely miscible with water, alcohols, acetone, and epoxy resins.

Acetonitrile Benefits

Resins have excellent cutting quality and beam stability, phospholipids in acetonitrile limits the loss of membrane lipids.

Dehydrating Agent for SEM

Also used as a dehydrating agent for cells prepared for Scanning Electron Microscopy (SEM).

General Solvent Properties

Miscible in all proportions with water, ether, chloroform, acetone, and the hydrocarbons xylene, toluene, and benzene, rapid without excessive shrinkage and hardening, low toxicity; low fire and explosion hazard.

General Solvent Disadvantages

Odorous - should be used in well-ventilated room, evaporates rapidly, dyes are not soluble in tetrahydrofuran.