Dehydration

Dehydration

Process of removing intercellular and extracellular water from the tissue following fixation and prior to wax impregnation

Dehydrating agents

Solutions utilized in dehydration

False (30%)

True or false: dehydration starting with 40% ethanol is recommended in embryonic tissues

1. Alcohol

2. Acetone

3. Dioxane 4 - cellosolve

4. Triethyl phosphate

5. Tetrahydrofuran

Five commonly used dehydrating agents: (AlAc DiTTe)

Ethyl alcohol (ethanol)

Alcohol recommended for routine duration of tissues; a clear colorless flammable fluid; beat dehydrating agent

Methyl alcohol

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

Butyl alcohol

Slow dehydrating agent; utilized in plant and animal microtechniques; recommended for tissues which do not require rapid processing

37° C

The temperature that will hasten dehydration time

Anhydrous copper sulfate

It is placed in the bottom of the container and covered with filter paper to insure complete dehydration

Blue

Discoloration of copper sulfate crystals that will indicate full saturation of their dehydrating fluids with water

Acetone

A cheap, rapid-acting the hydrating agent utilized for most urgent biopsies which is dehydrated in 1/2 to 2 hours; more miscible with epoxy resins; its use has been limited only to small pieces of tissues

Dioxane (Diethylene dioxide)

Excellent dehydrating and clearing agent readily miscible in water, melted paraffin, alcohol and xylol; produces less tissues shrinkage; expensive and extremely dangerous

Graupner's method

Method used as a time schedule for dehydration with dioxane

Weiseberger's method

Another method in dehydration with dioxane; tissue is wrapped in a gauze bag and suspended in a bottle containing dioxane and a little anhydrous calcium oxide; 3-24 hrs

Cellosolve (Ethylene glycol monoethyl ether)

Dehydrates rapidly; combustible at 110 to 120° F; toxic by inhalation skin contact and ingestion

1. Reproductive

2. Fetal

3. Urinary

4. Blood

Systems that are particularly vulnerable to the toxic side effects of cellosolve following exposure (4): FRUB

Propylene-based glycol ethers

Should be used instead of ethylene-based glycol ethers

Triethyl phosphate

Removes water readily and produces very little distortion and hardening of tissue; soluble in alcohol water, ether, benzene, chloroform, acetone and xylene

Tetrahydrofuran (THF)

Reagent that both dehydrates and clears tissues; used for demixing, clearing and dehydrating paraffin sections before and after staining; does not dissolve out aniline dyes; toxic if ingested or inhaled

Phenol (4%)

Added to each of the 95% ethanol baths; acts as a softener for hard tissues

Molliflex (glycerol/alcohol mixture)

Where hard tissues are immersed in

Ascending grades

Grades of dehydration

70%

Initial concentration:

-For routine tissues

30%

Initial concentration:

-For delicate tissues

10:1

Ratio of dehydrating agent to tissue

Ethanol (ethyl alcohol)

Recommended for routine tissue dehydration

Ethanol (ethyl alcohol)

Best dehydrating agent

Ethanol (ethyl alcohol)

Clear, colorless, flammable fluid

Fast acting, mixes with water and inorganic solvents

Methanol

Primarily for blood, and tissue films

Methanol

Toxic: blindness (if ingested)

Butanol

Utilized in plant and animal microtechniques

Macerate

Effect of dehydration:

Less than 70% conc

Methanol

Toxic: blindness (if ingested)

Butanol

Utilized in plant and animal microtechniques

Macerate

Effect of dehydration:

Less than 70% conc

Interfere staining

Effect of dehydration:

Longer storage using 70-80% alcohol

37 C

Temp that hastens dehydration

Anhydrous copper sulfate

Insures complete dehydration

Anhydrous copper sulfate

Accelerates dehydration by removal of water from the dehydrant

Blue

Color produced indicating presence of water (Anhydrous copper sulfate)

Acetone

For most urgent biopsies

Lipids are removed

Dioxane (diethylene dioxide)

Dehydrating and clearing agent

Miscible with paraffin, alcohol, xylene

Dioxane (diethylene dioxide)

Disadvantages:

-Tissues tend to ribbon completely

-Expensive

-Dangerous, highly toxic

Graupner's

Methods for dioxane dehydration:

-uses pure dioxane and paraffin

Weiseberger's

Methods for dioxane dehydration:

-Tissue is wrapped in gauze and suspended in a battle containing dioxane and anhydrous calcium oxide/quicklime

Cellosolve

Ethylene glycol monoethyl ether

Cellosolve

Combustible at 110-120F

Toxic

Triethyl phosphate

Removes water very readily and produce very little distortion

Minimum shrinkage

Tetrahydrofuran

Dehydrating and clearing agent

May be used for demixing, clearing, and dehydrating paraffin sections

Tetrahydrofuran

Dehydrating agent that gives improved staining results

Tetrahydrofuran

Has an offensive odor

Cause conjunctival irritation