 Process of removing intercellular and

extracellular water from the tissue following

fixation and prior to wax impregnation

Characteristics of an Ideal Dehydrating Solution

1. Should dehydrate rapidly without producing
considerable shrinkage or distortion of tissues
2. Should not evaporate very fast
3. Should be able to dehydrate even fatty tissues
4. Should not harden tissues excessively
5. Should not remove stains
6. Should not be toxic to the body
7. Should not be a fire hazard
Commonly used Dehydrating Agents:
1. Alcohol
2. Acetone
3. Dioxane
4. Cellosolve
5. Triethylphosphate
6. Tetrahydrofuran
I. ALCOHOL
 Ethyl alcohol (ethanol) – recommended
for routine dehydration
- clear, colorless, flammable fluid
- Best dehydrating agent
(fast acting, mixes with water and many
organic solvents, penetrates tissues easily)
- Not poisonous
- Not very expensive
 Methyl alcohol – for blood and tissue films;
smear preparations
- Toxic dehydrating agent
 Butyl alcohol – utilized in plant and
animal micro-techniques
- Slow dehydrating agent – produces less
shrinkage and hardening than ethanol
- recommended for tissues
which do not require rapid processing
 Tissues is passed through a series of
progressively increasing concentrations of
alcohol
 Temperature of 37°C – hasten dehydration
time
 For complete dehydration: ¼ in deep of
anhydrous copper sulfate is placed at the
bottom of the container and covered with
filter paper (accelerate dehydration by
removing water from dehydrating fluid)
- Blue discoloration of copper sulfate
crystals will indicate full saturation of
dehydrating fluids with water
- Alcohol is discarded and changed with a
fresh solution
ACETONE
 Cheap
 Rapid acting dehydrating agent (dehydrates in
½ to 2 hours)
- Penetrates tissues poorly
 Clear, colorless fluid – mixes with water,
ethanol and most organic solvents
 More miscible with epoxy resins than alcohol
 Highly flammable – requires considerable care
in handling
 Causes brittleness in tissues if placed for
prolonged period of time
 Most lipids are removed from tissues
 Use is limited to small pieces of tissues
due to extreme volatility and
inflammability
 Not recommended for routine dehydration
purposes – due to tissue shrinkage
 Excellent dehydrating and clearing agent
 Readily miscible in water, melted paraffin,
alcohol and xylol
 Produces less tissue shrinkage compared to
alcohol dehydration
 Tissues can be left for long period of time
without affecting the consistency or staining
properties
 Tend to ribbon poorly
 Expensive
 Extremely dangerous (main disadvantage)
- Vapor produces a cumulative and highly toxic
action in man
- Not recycled as risk of creating explosive
peroxides increases
 Two methods of dehydration :
- Graupner’s Method:
1st pure dioxane soln --------------------- 1 hour
2nd pure dioxane soln -------------------- 1 hour
3rd pure dioxane soln -------------------- 2 hours
1st paraffin wax ---------------------------- 15 minutes
2nd paraffin wax --------------------------- 45 minutes
3rd paraffin wax --------------------------- 2 hours
Embed in mold and cool in water
- Weiseberger’s Method – tissue is wrapped in a
gauze and suspended in a bottle containing dioxane
and a little anhydrous calcium oxide
- water is displaced from the tissue and absorbed
by calcium oxide or quick lime
- dehydration period : 3 – 24 hours
(ETHYLENE GLYCOL MONOETHYL ETHER)
 Dehydrates rapidly
 Tissue may be transferred from water or
normal saline to cellosolve and store it for
months without hardening or distortion
 Ethylene glycol ether – combustible at 110 –
120°F
- Toxic by inhalation, skin contact and
ingestion
- Following exposure, the reproductive ,fetal,
urinary and blood systems are particularly
vulnerable to toxic side effects
 Removes water very readily and produces
very little distortion and hardening of
tissue
 Soluble in water, alcohol, ether, benzene,
chloroform, acetone and xylene
 Dehydrate sections and smears following
certain stains and produces minimum
shrinkage
 Both dehydrates and clears tissues
 Miscible in both water and paraffin
 Dissolve may substances including fats
 Miscible in lower alcohols, ether, chloroform,
acetone, benzene, and xylene
 Used for demixing, clearing and dehydrating
paraffin sections before and after staining
 Causes less shrinkage and easier cutting of
sections with fewer artifacts
 Does not dissolve aniline dyes
 Toxic if ingested or inhaled
- Vapors cause nausea, dizziness, headache
- Eye and skin irritant – prolonged exposure
(6 mons) cause conjuctival irritation
- no practical way of absolutely
protecting the skin against contact – use
of THF should be avoided
Additives to Dehydrating Agents:
- 4% phenol added to 95% ethanol baths –
acts as softener for hard tissues
- Immersed in glycerol/alcohol mixture
- Molliflex