MIDTERM LEC: DEHYDRATION Flashcards
Follows fixation, and decalcification (if applicable)
DEHYDRATION
It is the removal of water from tissue in preparation for impregnation
DEHYDRATION
Since most fixatives are aqueous solutions, placing the fixed tissue in molten paraffin will not achieve impregnation because paraffin wax and water (from fixative) do not mix. Hence, dehydration must be done.
DEHYDRATION
Done as brief as possible and at a tissue-to-fixative ratio of 1:10
DEHYDRATION
Common Dehydrating Agents:
I. Alcohol
II. Acetone
III. Dioxane
IV. Cellosolve
V. Triethyl phosphate
VI. Tetrahydrofuran
Characteristics of Ideal Dehydrating Agents:
- Rapid action, with minimal tissue shrinkage and distortion
- Should not evaporate fast
- Able to dehydrate even fatty tissues
- Should not harden tissues excessively
- Should not remove stains
- Non-toxic, and not a fire hazard
Done in ascending grades to avoid distortion of tissue: 70% ROH -> 90% ROH ->100% ROH
ALCOHOLS
For delicate tissues, start at 30%
ALCOHOLS
ALCOHOLS
Initial alcohol conc. depends on the size and nature of tissue and the fixative used:
Strong initial conc. shrinkage and brittleness
Prolonged dehydration in less than 70% ROH tissue maceration
ALCOHOLS
37C
will hasten dehydration rate (for urgent exams)
ALCOHOLS
To ensure complete dehydration:
Add atleast ¼ deep layer of anhydrous Cu2SO4 at bottom of container, and cover with filter paper
ALCOHOLS
indicates full saturation of dehydrating fluids with water, thus alcohol must be changed with
a fresh solution
Bluing of copper sulfate crystals
ALCOHOLS:
- Ethyl alcohol (Ethanol)
- Methanol
- Butanol
Best dehydrant because it is fast-acting, mixes with water and many organic solvents, and penetrates tissues easily
Ethyl alcohol (Ethanol)
Not poisonous and not very expensive
Ethyl alcohol (Ethanol)
Clear, colorless, flammable
Ethyl alcohol (Ethanol)
For blood & tissue films, smears
Methanol
Plant and animal microtechniques
Butanol
Less tissue shrinkage and hardening but slow
Butanol
Fastest dehydrant (1/2 to 2 hours), thus for rapid biopsies
ACETONE
Clear, colorless, and more miscible with epoxy resins than alcohol
ACETONE
DADV: Flammable, extremely volatile, and not recommended for routine work because of considerable shrinkage and brittleness
ACETONE
Lipids are removed from tissue
ACETONE
Both a dehydrant and clearing agent
DIOXANE (DIETHYLENE DIOXIDE)
ADV: Less tissue shrinkage, prolongation is possible
DIOXANE (DIETHYLENE DIOXIDE)
DADV: extremely dangerous because of toxicity, and risk of explosion; expensive, tissues will have poor ribbons
DIOXANE (DIETHYLENE DIOXIDE)
DIOXANE (DIETHYLENE DIOXIDE) Methods:
- Graupner’s
- Weiseberger’s
pure dioxane -> paraffin
Graupner’s
wrapping tissue in gauze and suspension
to bottle containing dioxane with anhydrous calcium oxide or quicklime
Weiseberger’s
DIOXANE (DIETHYLENE DIOXIDE)
Note: Tissues treated with chromic acid must be ____
thoroughly washed with water prior to dioxane treatment
ADV: no shrinkage in prolongation
CELLOSOLVE/ETHYLENE GLYCOL MONOGLYCOL ETHER
DADV: Combustible at 110-120F, toxic to reproductive, fetal, urinary, and blood systems
CELLOSOLVE/ETHYLENE GLYCOL MONOGLYCOL ETHER
If cannot be avoided, propylene-based glycol ethers should be used instead of ethylene-based
CELLOSOLVE/ETHYLENE GLYCOL MONOGLYCOL ETHER
A:rapid, little distortion and hardening
TRIETHYL PHOSPHATE
Both a dehydrant and clearing agent
TETRAHYDROFURAN
Dissolves fats
TETRAHYDROFURAN
Most staining procedures give improved results with THF
TETRAHYDROFURAN
Has rather offensive odor, thus room must be well-ventilated
TETRAHYDROFURAN
Toxic to eyes (conjunctival irritation) and skin (Teflon gloves may be used, but it is recommended to avoid THF use)
TETRAHYDROFURAN
ADDITIVES:
- 4% phenol
- Molliflex (glycerol alcohol mixture)
added to 95% ETOH; softener for hard tissues
4% phenol
softener
Molliflex (glycerol alcohol mixture)
