DEHYDRATION

Medical Technology
College of Allied Medical Professions
Daniel Mercado Medical Center – Institute of Health Sciences
DEHYDRATION
• PROCESS OF REMOVING INTERCELLULAR
AND EXTRACELLULAR WATER FROM TISSUE
FOLLOWING FIXATION AND PRIOR TO WAX
IMPREGNATION.
 DEHYDRATING AGENTS
• SOLUTIONS UTILIZED TO MAKE DEHYDRATION
POSSIBLE.
• MANY OF THESE DEHYDRATING AGENTS ARE
ALCOHOLS.
• USED TO REMOVE AQUEOUS TISSUE FLUIDS
WITH LITTLE DISRUPTION TO THE TISSUE CAUSED
BY DIFFUSION CURRENTS.
 DRYING VS. DEHYDRATION

DRYING DEHYDRATION
• The removal of water by • Involves slow substitution
evaporation from a solid, of the water in the tissue
semi-solid or liquid. with an organic solvent.

TAKE NOTE : Solid Tissues should NEVER be allowed to air dry.

 TAKE NOTE!

• Whatever dehydrating agent used, the

amount in each step should not be less
than 10 minutes the volume of the
tissue in order to ensure complete
penetration of the tissue by dehydrating
solution.
 TAKE NOTE!

• It is also important to keep the

dehydration times as brief as possible
to minimize the risk of extracting
cellular constituents .
 DID YOU KNOW?

• Almost any water miscible ,

Anhydrous fluid can be used as a
dehydrating agent.
CHARACTERISTICS OF AN IDEAL DEHYDRATING
SOLUTION
• It should dehydrate rapidly without
producing considerable shrinkage or
distortion of tissues.
• It should not evaporate very fast.
• It should not be able to dehydrate even fatty
tissues.
• It should not harden tissues excessively.
CHARACTERISTICS OF AN IDEAL DEHYDRATING SOLUTION

• It should not remove stains.

• It should not be toxic to the body.
• It should not be a fire hazard.
COMMONLY USED DEHYDRATING AGENTS
• Alcohol (most common)
• Acetone
• Dioxane
• Cellusolve
• Triethyl phosphate
• Tetrahydrofuran
 Take Note !
• It is not advisable to transfer fixed tissues
directly from water or aqueous fixative
directly into absolute ethanol.

• Doing so can cause a rapid removal of

water which can distort the appearance
of more delicate cells and structures.
 Take Note !
• It is advisable to remove water gently and
allow the tissue to slowly adjust to its
removal.

• The more delicate the tissue , the more

gently this should be done , but there is no
hard and fast rule.
ETHYL ALCOHOL (ethanol)
• alcohol recommended for routine
dehydration of tissues

• It is a clear, colorless , flammable fluid.

• Best dehydrating agent because it is fast

acting .
METHYL ALCOHOL

• Toxic dehydrating agent

• Primarily employed for blood and

tissue films and for smear preparation
BUTYL ALCOHOL
• Utilized in plant and animal micro-techniques

• A slow dehydrating agent producing less

shrinkage and hardening than ethyl alcohol

• Recommended for tissues which do not

require rapid processing
CLEARING

• Property of the solvents used –when

they have a relatively high refractive
index and when tissue is immersed in
them , the tissue becomes
transparent and CLEAR.
 Take Note !
• Never leave tissues in 95% or 100%
ethanol more than a total of 2 hours or
the tissues will harden.

• Tissues can be stored in 70% ethanol at

any time during an interruption in the
routine.
 General Schedule for Alcohol Dehydration of Big
Tissues According to Type of Fixative Used:
Susa, Carnor or
10% Formol- Zenker or Helly’s Bouin’s Fluid Formol Flemmings FLuid
saline Sublimate

Running Water 1-12 1-12

Alcohol 30% 1-6 ½-3
Alcohol 50% 1-6 ½-3
Alcohol 70% 3-12 1-6 3-12 ½-3
Alcohol 90% 3-12 1-6 3-12 1-6 1-3
Absolute Alcohol (1) 1-2 1-2 1-2 1-2 1-2
Absolute Alcohol (1) 1-2 1-2 1-2 1-2 1-2
Absolute Alcohol (3) 1-2 1-2 1-2 1-2 1-2
A Typical Dehydration Sequence for Specimens Not
More Than 4mm Thick Would Be:

70% ethanol 15 min.

90% ethanol 15 min.
100% ethanol 15 min.
100% ethanol 15 min.
100% ethanol 30 min.
100% ethanol 45 min.
 Take Note !

• A temperature of 37°C will hasten

dehydration time and is specially used
for tissue sections that require urgent
examinations such as fragmentary
biopsies.
 Take Note !
• A layer of anhydrous copper sulfate, about ¼
inch deep is placed in the bottom of the
container and covered with filter paper. This
will accelerate the dehydration by removing
water from the dehydrating fluid.

BLUE DISCOLORATION OF COPPER SULFATE CRYSTAL = Full Saturation of

dehydrating fluids with water
 ETHANOL (ethyl alcohol)
Advantages Disadvantages
• Nontoxic • Expensive
• Miscible in all proportions with • Long periods in absolute ethanol
water will cause excessive shrinkage and
hardening
• Little shrinkage if graded alcohols • May be difficult to obtain
are used

• Can be used on eyes and embryos , • May have prohibitive taxes that
if graded alcohols are used necessitate troublesome book
keeping
 ETHANOL (ethyl alcohol)
Advantages Disadvantages
• Fast acting • Extracts methylene blue and other
thiazine dyes from sections
• Still considered best dehydrating solution • Extracts more lipids than acetone

• Reliable • May cause more shrinkage of specimen

• Appears to cause less extraction of • May react with unreduced 0s04

cellular components in general than other remaining in specimen
agents

• Only slightly miscible with most resins

 BUTANOL (butyl alcohol)
Advantages Disadvantages
• Less shrinkage and hardening • Odorous
than with ethyl
• Excellent for slow processing • Slow-acting

• Miscible with paraffin • Long periods of infiltration

necessary
• Dehydrating power low
 TERTIARY BUTANOL (butyl alcohol)
Advantages Disadvantages
• Universal solvent- acts as • Odorous
dehydrating and clearing agent
• May be used in staining series as a • More expensive than butanol
dehydrating agent
• Mixes with water , ethanol , xylene • Primary infiltration must be done in
and paraffin in all half tertiary butanol and half
paraffin, prior to paraffin
impregnation
• Reagent tends to solidify at room
temperature or below 25°C
 ISOPROPANOL (isopropyl alcohol)
Advantages Disadvantages
• Excellent substitute for ethanol • Cannot be used in the celloidin
technique since nitrocellulose is
insoluble in it
• Less shrinkage and hardening than • Cannot be use for preparing staining
ethanol solutions, since dyes are not soluble
in it
• No government restrictions on its
use
• Sufficiently water-free to use in place
of absolute ethanol
• Lillie considers it “the best all-around
substitute for ethyl alcohol
• Less expensive than tax-free alcohol
 PENTANOL (amyl alcohol)
Advantages Disadvantages
• Miscible with 90% alcohol , • Toxic
toluene and xylene
• Dissolves paraffin wax • Cannot be used in poorly
ventilated room
• Not miscible with water
 ALCOHOL BOILING POINT
Ethanol (ethyl alcohol) 78.3 °C

Butanol (butyl alcohol) 117.7 °C

Tertiary butanol (butyl alcohol) 82.8 °C

Isopropanol (isopropyl alcohol) 82.3 °C

Pentanol (amyl alcohol) 215 °C

ACETONE
• IS A cheap, rapid-acting dehydrating
agent

• Utilized for most urgent biopsies which it

dehydrates in 1/2 to 2 hours
ACETONE

• Most lipids are removed from tissues

with this dehydrating agent.

• Not recommended for routine

dehydration purposes.
 Advantages Disadvantages
• Rapid dehydrating agent • Requires clearing agents

• Less expensive than ethanol • Volume must be 20 times that of the tissue
• Does not extract methylene blue and other • Best processing requires a graded series of
dyes from stained sections a mixture of acetone and xylene before one
can go into paraffin
• May cause less shrinkage of specimen than • Needs good ventilation
ethanol
• Not reactive with 0S04 remaining in • Evaporates rapidly
specimen
• Miscible with most embedding resins • Flammable

• Absolute acetone is easily contaminated

with water, resulting in complete
dehydration
• Uranyl acetate and phosphotungstic acid
are only soluble in dilute solutions of
acetone
DIOXANE ( Diethyl Dioxide)
• IT produces less tissue shrinkage as compared
to alcohol dehydration.

• Tissues can be left in this reagent for long

periods of time without affecting the
consistency or staining properties of the
specimen
DIOXANE ( Diethyl Dioxide)

• Tissues may be placed directly into the

solution after washing out because dioxane is
miscible with water and paraffin.

• Tissue sections dehydrated with dioxane tend

to ribbon poorly.
Refractive Index of 1.42
Time Schedule for Dehydration With Dioxane
(Graupner’s Method)

(1st) Pure Dioxane Solution 1 hour

(2nd) Pure Dioxane Solution 1 hours
(3rd) Pure Dioxane Solution 2 hours
(1st) Paraffin wax 15 mins.
(2nd) Paraffin wax 45 mins.
(3rd) Paraffin wax 2 hours
Embed in mold and cool in water
DIOXANE ( Diethyl Dioxide)
• In Weiseberger’s method, the tissue is wrapped in
gauze bad and suspended in a bottle containing
dioxane and a little anhydrous calcium oxide.

• Water is displaced from the tissue by dioxane and

in turn absorbed by calcium oxide or quicklime.

DEHYDRATION PERIOD : 3-24 hours

 TAKE NOTE!
• DIOXANE vapor produces a cumulative and
highly toxic action in man; hence, it should
not be used routinely.

• It should not be recycled as the risk of

creating explosive peroxidases increases
greatly.
 TAKE NOTE!

• Tissues which have been treated with

a chromate fixative , e.g. regaud’s or
mollers fluid, should be thoroughly
washed in running tap water prior to
treatment with dioxane in order to
remove the chromate.
 Advantages Disadvantages
• Universal solvent- it dehydrates and • Needs large volume for dehydration
clears
• Miscible with water, alcohol, xylene • Costs about four times more than
and paraffin does absolute alcohol
• Does not harm tissue over long time • Must be used in well-ventilated
periods rooms
• Faster dehydrant than ethanol • Cumulatively toxic

• Odorous

• Distorts tissue-tissue containing

cavities
CELLUSOLVE ( Ethylene Glycol
Monoethyl ether)
• Dehydrates rapidly

• The tissue may be transferred from water

or normal saline directly to cellusolve
and stored in it for months without
producing hardening or distortion.
CELLUSOLVE ( Ethylene Glycol
Monoethyl ether)
• Are toxic by inhalation, skin contact and
ingestion.

CAUTION: Ethylene glycol ethers are

combustible at 110-120°F
CELLUSOLVE ( Ethylene Glycol Monoethyl
ether)
• Following exposure , the reproductive, fetal,
urinary and blood systems are particularly
vulnerable to their toxic side effects.

• If it cannot be avoided, propylene-based

glycol ethers should be used instead of
ethylene-based glycol ethers.
 Advantages Disadvantages
• Rapid dehydrating agent • Expensive

• Tissue may remain in it for • Rapidly absorbs water

months without injury from the air

• Avoids distortion and does • Requires clearing agent

not require graded
solutions
TRIETHYLPHOSPHATE
• It removes water very readily and
produces very little distortion and
hardening of the tissue

• It is soluble in alcohol, water, ether,

benzene, chloroform, acetone and xylene
TRIETHYLPHOSPHATE

• Used to dehydrate sections and

smears following certain stains and
produces minimum shrinkages.
 Advantages Disadvantages
• May be used in routine paraffin • None
technic
• Displaces water readily with slight
distortion

• Does not harden tissue excessively

• May be used as a dehydrating

solution in the staining sequence

• Soluble in alcohols , benzene ,

toluene , xylene, ether, chloroform
TETRAHYDROFURAN (THF)
• A reagent that both dehydrates and clears
tissues since it is miscible in both water and
paraffin

• Used for demixing , Clearing and

dehydrating paraffin sections before and
after staining.
TETRAHYDROFURAN (THF)
• It causes less shrinkage and easier cutting
of sections with fewer artifacts

• Most staining procedures give improved

results with tetrahydrofuran
TETRAHYDROFURAN (THF)

• THF is toxic if ingested or inhaled

• Vapor causes nausea, dizziness,

headache and anesthesisa.
TETRAHYDROFURAN (THF)
• It is an eye and skin irritant , and
prolonged exposure (up to 6 months)
may cause conjunctival irritation.

• Should be done in a well ventilated

room
TETRAHYDROFURAN (THF)

• Although teflon gloves may be

suitable , THF SHOULD BE AVOIDED IF
POSSIBLE, AS there is no practical way
to absolutely protect skin against
contact.
 Advantages Disadvantages
• Miscible in all proportions with • Odorous – should be used in well
water, ether, chloroform, acetone, ventilated room
and hydrocarbons xylene, toluene
and benzene
• Rapid without excessive • Evaporates rapidly
shrinkage and hardening
• Low toxicity; low fire and • Dyes are not soluble in
explosion hazard tetrahydrofuran
• Not toxic
• Better results than most
universal solvents
• Solvents of mounting media
 DEHYDRATING AGENT BOILING POINT
Acetone 56 °C

Dioxane (Diethylene 101.5 °C

dioxide)
Cellusolve (Ethylene 156.4 °C
glycol monoethyl esther)
Triethylphosphate 215 °C
 DEHYDRATING AGENTS FOR
ELECTRON MICROSCOPY
• Tissue processing for transmission
electron microscopy (TEM) is
commonly accomplished using
ethanol as a dehydrating solvent and
propylene oxide as a transition fluid.
 DEHYDRATING AGENTS FOR ELECTRON
MICROSCOPY
Ethanol
• Solubilizes lipids

Propylene oxide
• Completely miscible with embedding resins
• Can infiltrate tissues readily and reduce the
viscosity of embedding resin mixtures
 DEHYDRATING AGENTS FOR
ELECTRON MICROSCOPY
Acetonitrile
• is a good substitute for propylene oxide.

• Non-carcinogenic, less toxic and not as flammable

as propylene oxide

• Also used as a dehydrating agent for cells

prepared for Scanning electron microscope (SEM)
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