DEHYDRATION - Histopathologic Techniques

Medtech

1. DEHYDRATION

2. OBJECTIVES
•Describe purpose of dehydration
•Enumerate the different dehydrating agents
•Describe their advantages and disadvantages

3. Principle:

•Removal of intercellular and extracellular water from the tissue
done after fixation and replace them with dehydrating agents
•Done after fixation and after bones and teeth have been
decalcified

4. Considerations
•Dehydration is done by placing fixed specimen in serially increasing
strengths of alcohol (70%, 95%, ethyl alcohol in water and 100% ethyl
alcohol respectively)
•This serial immersions remove water gradually to prevent disruptions in the
tissue due to diffusion currents (shrinkage and hardening leading to
distortion)
•Delicate tissues like embryonic tissues usually start with 30% ethyl alcohol in
water
•95% alcohols that harden the surface only with poor penetration will cause
unequal impregnation with consequent poor cutting of sections

5. Considerations
•Dehydration times should be as brief as possible to minimize the risk
of extracting cellular constituents
•Prolonged storage in alcohol causes maceration, especially at lower strengths
below 70%
•Storage of specimen may be done at 70-80% alcohol but not for
extended periods due to interference in their staining properties
•Dehydrating agent must not be less than 10x the volume of the tissue for
proper penetration

6. Considerations
•For urgent examinations, temperature may be increased to 37°C
•For complete dehydration with alcohol:
•A layer of anhydrous copper sulfate about ¼ inch is placed at the bottom of
the container and covered with filter paper
•Accelerates dehydration by removing water from dehydrating fluid
•Blue discoloration of copper sulfate crystals indicate full saturation of
dehydrating fluids with water- alcohol should be discarded and changed with
a fresh solutioin

7. Commonly Used Dehydrating Agents
1.Alcohol (most common)
2.Acetone
3.Dioxane 4-cellosolve
4.Triethyl phosphate
5.Tetrahydrofuran

8. A. Alcohol
•used in increasing concentration (70% to 95% ) to prevent
shrinkage and hardening of tissue which may lead to distortion.
•Note:
•Too low alcohol concentration: leads to tissue maceration
•concentrated alcohol: tissue hardening

9. A. Alcohol
1.Ethyl alcohol
•best dehydrating agent (fast acting, mixes with water and
penetrates tissues easily)
•Clear colorless, flammable
• Most recommended for routine dehydration
2.Methyl alcohol
•toxic
•used for blood and tissue films and for smear preparations

10. A. Alcohol
3. Butyl alcohol
•Utilized in plant and animal microtechniques
•Slow acting causing less shrinkage and hardening than ethyl
alcohol
•Recommended for tissues which do nor require rapid processing

11. B. Acetone
•Cheap and rapid acting (dehydrates biopsies for ½ to 2 hours)
but penetrates poorly
•Clear colorless fluid that mixes with water, ethanol and most
organic solvents
•More miscible with epoxy resins than alcohol but extremely
volatile and flammable
•Removes most lipids from tissues
•not recommended for routine tissue purposes due to
considerable tissue shrinkage

12. C. Dioxane (Diethylene dioxide)
•excellent dehydrating and clearing agent
•miscible with water, paraffin , alcohol and xylol
•Less tissue shrinkage vs alcohol
•Tissues may be stored for long periods without loss in consistency or
staining properties
•Disadvantages: expensive, toxic vapour, tissues tend to ribbon poorly
•Not routine due to highly toxic effect (use only in well ventilated room)
•Should not be recycled due to the risk of forming explosive peroxides

13. D. Cellosolve (Ethylene glycol
monoethyl ether)
•rapid dehydrating agent,
•Specimen may be transferred from water or normal saline
directly into cellosolve for storage with no hardening or
distortion
•Flammable at 43-49°C, toxic by inhalation, skin contact
and ingestion to the reproductive, fetal, urinary and
blood (Substitute: Propylene based ethylene glycos)

14. D. Triethyl phosphate
•soluble in water, alcohol, ether, benzene chloroform,
acetone and xylene
•removes water from tissues readily with little distortion
and hardening of tissues.

15. D. Tetrahydrofuran
•clearing and dehydrating agent since it is miscible in both water and
paraffin.
•causes less shrinkage, easier cutting of sections with fewer artifacts
•Improves most staining procedures
•Toxic on ingestion and inhalation, offensive odor (use in well
ventilated room)
•Eye irritant- prolonged exposure (up to 6 mos) may cause
conjunctival irritation
•Skin irritant- no practical way to protect skin except for Teflon
gloves