Dehydration- MIDTERMS L9

DEHYDRATION

• Aim to remove fixative and water from the tissue and replacing them with dehydrating fluid in preparation for impregnation.

• Dehydrating fluids are generally used in increasing strengths.

• Removal of intracellular and extracellular water from the tissue following fixation and prior to wax impregnation. It is important to keep the dehydration times as brief as possible to minimize the risk of extracting cellular constituents.

DRYING

Removal of water by evaporation

DEHYDRATION

Slow substitution of water in the tissue with organic solvent

INCREASING STRENGTHS

All the aqueous tissue fluids are removed but with little disruption to the tissue due to diffusion currents.

DEHYDRATION FOR DELICATE TISSUES

Particularly embryonic and animal tissues, it is recommended to start processing with 30% ethyl alcohol.

CHRACTERISTICS OF AN IDEAL DEHYDRATING SOLUTION

• It must dehydrate rapidly

• Not evaporate very fast

• Be able to dehydrate fatty tissues

• It should not harden the tissue

• It should not remove stains

• Not toxic

• Not be a fire hazard

COMMONLY USED DEHYDATING AGENTS

• Alcohol (Ethyl alcohol, methyl alcohol, Butyl alcohol)

• Acetone

• Dioxane

• Cellosolve

• Triethyl Phosphate

• Tetrahydrofuran

ALCOHOL

Tissue is passed through a series of progressively increasing concentrations of alcohol.

80-95% ALCOHOL

Liable to produce considerable shrinkage and hardening of tissues leading to distortion

95% OR ABSOLUTE ALCOHOL

Tend to harden only the surface of the tissue while the deeper parts are not completely penetrated

PROLONGED STORAGE BELOW 70% ALCOHOL

Tend to macerate the tissue

ETHYL ALCOHOL

Recommended for routine dehydration. Clear, colorless flammable fluid. Considered to be the best dehydrating agent because it is fast-acting.

METHYL ALCOHOL

Toxic dehydrating agent; used for blood and tissue films and for smear preparations

BUTYL ALCOHOL

• Utilized in plant and animal micro techniques; slow dehydrating agent, produces less shrinkage and hardening. Recommended for tissues who do not require rapid processing.

• Not for urgent

GENERAL SCHEDULE FOR ALCOHOL DEHYDRATION

• 70 % alcohol- 6 hrs.

• 90 % alcohol- 12 hrs.

• 100% (1st concentration)- 2 hrs.

• 100% (2nd concentration)- 1 hr.

• 100% (3rd concentration)- 1 hr.

ACETONE

• Both fixative and dehydrating agent.

• Cheap, rapid acting dehydrating agent. Dehydrates in ½ to 2 hours.

• Clear, colorless highly flammable and extremely volatile fluid.

• Rapid in action but penetrates tissues poorly and causes brittleness in tissues that are prolonged dehydrated.

• Considerable tissue shrinkage

• Not recommended for routine dehydration

DIOXANE (DIETHYLENE OXIDE)

• Excellent dehydrating and clearing agent

• Produces less tissue shrinkage

• Tissues can be left for long periods of time without affecting the consistency or staining properties of the specimen

• Tissue sections dehydrated with dioxane tend to ribbon poorly

• Expensive and extremely dangerous

CELLOSOLVE (ETHYLENE GLYCOL MONOETHYL ETHER)

• Both dehydrating and clearing agent

• Dehydrates rapidly.

• The tissue may be transferred from water or normal saline directly to cellosolve and stored in it for months without producing hardening or distortion.

• Ethylene glycol ether is combustible at 110°F to 120°F and is toxic.

• Propylene based glycol ether should be used instead.

TRIETHYL PHOSPHATE

• It removes water very readily and produces very little distortion and hardening of tissue

• It is used to dehydrate sections and smears following certain stains and produces minimum shrinkage

TETRAHYDROFURAN

• It both dehydrates and clears tissues since it is miscible in both water and paraffin.

• It may be used for demixing, clearing and dehydrating paraffin sections before and after staining.

• It causes less shrinkage and easier cutting of sections with fewer artifacts.

• Do not dissolve aniline dyes

• Toxic if ingested or inhaled

ADDITIVES TO DEHYDRATING AGENT S

• 4% phenol + each 95% ETOH baths

• Anhydrous copper sulfate

4% PHENOL + EACH 95% ETOH BATHS

Acts as a tissue softener for hard tissues such as tendons, nails, or dense fibrous tissues.

ANHYDROUS COPPER SULFATE

• Can act as both as a dehydrating agent and an indicator of water content of the last bath (100% ETOH).

• To ensure complete dehydration, a layer of anhydrous copper sulfate, about 1/4-inch deep is placed in the bottom of the container and covered with filter paper.

• BLUE COLOR OF COPPER SULFATE CYSTALS

• FULL SATURATION→ DISCOLORATION

DEHYDRATION TEMPERATURE

Room Temperature (18-30*C)

DECALCIFICATION TEMPERATURE

37*C