DEHYDRATION

OBJECTIVES
• Describe purpose of dehydration
• Enumerate the different dehydrating agents
• Describe their advantages and disadvantages
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
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
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
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
Commonly Used Dehydrating Agents
1. Alcohol (most common)
2. Acetone
3. Dioxane 4-cellosolve
4. Triethyl phosphate
5. Tetrahydrofuran
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
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
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
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
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
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)
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.
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