HISTOPATHOLOGY LEC
LECTURE 7: DEHYDRATION
PROF. FRANCIS IAN SALVER, RMT, MD
JUNE 23, 2021
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DEHYDRATION
● Process of removing extracellular and intracellular water
from the tissue following fixation and decalcification (for
bone and teeth) and prior to wax impregnation
● Done after the tissue is fixed and decalcified
● Done prior to wax impregnation
Step 1: Dehydration
● Remove first water from the tissue and replacing it with
alcohol
● Alcohol is acting as a dehydrating agent.
● After Step 1 – tissue is filled up with alcohol
● Main purpose of alcohol: penetrate the tissue and replace
the water
Step 2: Clearing
● Removal of the dehydrating agent (alcohol) from the tissue
replacing it with a clearing agent such as xylene
● After step 2 – tissue is filled up with clearing agent
○ Clearing agents are highly volatile and easily
evaporate with heat
Step 3: Impregnation
● Removal of the clearing agent and replacing it with melted
paraffin
○ Melted paraffin has high temperature which
causes the evaporation of the clearing agent
● Tissue is now is impregnated with wax
Tissue impregnated with paraffin wax
The purpose of the dehydration is to remove water since wax is
not miscible with water.
Wax impregnation will make easy for the medtech to do tissue
sectioning using a microtome
- For this to happen, paraffin must have completely infiltrated
the tissue and water should have been removed early in
dehydration
Review:
● Dehydration is simply the removal of water from
aqueous-fixed tissue.
● Since most fixatives are aqueous, this step is necessary to
prepare the tissue for embedding in nonaqueous media like
paraffin.
Dehydration
● Volume of dehydrating agent should be 10x than the tissue
● Many of the dehydrating agents are alcohols and are
generally used in increasing strength to remove water from
the tissue to avoid tissue distortion
● Dehydration starts by placing fixed specimen in 70% ethyl
alcohol in water then transferred into 95% ethyl alcohol
then into 100% ethyl alcohol
○ For delicate/soft tissue, dehydration can start as
low as in 30% ethyl alcohol
● Tissue should be immersed in an increasing concentration
of the dehydrating agent
● To avoid tissue distortions, we have to immerse the tissue
first in a lower concentration of alcohol then transfer it to
higher concentration
Why not immersed the tissue directly to 100% ethyl alcohol to
speed up processing in the laboratory?
● Grape in the left was dehydrated using increasing
concentration of alcohol
● Grape in the right was placed directly in 100% alcohol
● Which grape has lesser tissue distortion?
○ Left = immersed in an increasing concentration
● Placing tissues directly to concentrated dehydrating agent
will produce considerable shrinkage and hardening of
tissue leading to distortion
○ Also affect the quality of the thin section during
microtomy
● Concentrated alcohol only dehydrates the superficial layer
of the tissue while the deeper parts are poorly penetrated
○ The alcohol is penetrating the tissue and since it
is concentrated, it is causing the tissue to have
tissue distortions.
○ The lower portion or the inner portion will no
longer be penetrated by the concentrated alcohol
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 Characteristics of a Good Dehydrating Agent
● Dehydrate tissue rapidly without causing considerable
shrinkage or distortion of tissue
○ The pathologist might think that the tissue or cells
have undergone atrophy, when in fact the
decrease in size is brought about the dehydrating
agent
● Should not evaporate very fast (affects time of contact with
tissue)
○ If there is still water inside the tissue, there will be
a problem with wax impregnation
● Should not harden tissues excessively
● Should not cause the stain to be removed.
○ Apply stains to appreciate the microscopic details
● Should not be toxic to the body
○ In the lab, medtechs get to process 10 tissues a
day and they can't avoid the fact to use alcohol
(exposed to chemical) -= to not develop problems
later on dehydrating agents should not be toxic
● Should not be fire hazard
DEHYDRATING AGENTS
● Alcohol
● Acetone
● Dioxane
● Triethyl phosphate
● Tetrahydrofuran
Things to remember before performing dehydration
● In case of Carnoy’s fluid and other alcoholic fixatives, the
tissues are directly transferred to 90% or 95% or even
Absolute alcohol
○ Why tissues fixed in Carnoy’s fluid should not be
immersed in a lower concentration of Alcohol?
Because the tissue will most likely collapse
● Sometimes for soft tissues like an embryo, the first stage of
30% alcohol is used followed by a graded treatment of the
alcohol to prevent tissue shrinkage
● Tissues fixed in Heidenhan’s Susa fluid are transferred in
90% alcohol because lower grades of alcohol may cause
swelling of the fibrous tissues.
○ Immerse to 90%, then 95%, the 100%
○ If tissue is fixed in Susa’s fluid and placed in a
lower concentration of alcohol, there is a
probability of swelling especially on fibrous tissue
○ Affects appearance of tissue under microscope
Ethyl Alcohol
● Most commonly used dehydrating agent
● Recommended alcohol for routine dehydration procedure
○ Best dehydrating agent
● Clear, colorless and flammable fluid
● Fast acting
● Mixes well with water (it's easy to prepare 70 and 95%
concentrations)
○ You can easily prepare lower concentrations of
alcohol from absolute alcohol
● Penetrates the tissue well
○ Can remove water even in the innermost portion
of the tissue
○ To facilitate easy infiltration of wax during
impregnation step
● Not expensive!!
● No known toxic effect
● It may cause hardening, distortion and shrinkage of tissues
when left for longer period in ethanol or when immersed
directly to absolute ethanol
○ Affects cutting and appearance
Methyl Alcohol
● Both a dehydrating and fixative agent for blood and bone
marrow smear preparation
● Toxic dehydrating agent recommended for blood and tissue
for smear preparation
○ They do not affect the quality of stains used in
blood and bone marrow smears
● Methanol tends to harden tissues more than ethanol
(METigas) and is a poor lipid solvent; thus cannot be used
in organs with high lipid content (METaba)
● Highly volatile, highly flammable and expensive
○ Highly toxic to humans
● Can cause metabolic acidosis
Methanol metabolism
● Methanol if accidentally ingested will be acted upon by
alcohol dehydrogenase to formaldehyde
● Formaldehyde will be acted upon by formaldehyde
dehydrogenase converting it into formic acid
● As we convert, NAD is also converted to NADH
○ Body is losing NAD because for the process to
continue (from methanol to formic acid) NAD
needs to be consumed to form NADH
● Formic acid can make our blood acidic
○ Develops metabolic acidosis
● High levels of formic acid can also cause ocular toxicity
leading to blindness
● This process has to continuously happen, we need to
reconvert NADH to NAD by converting pyruvate to lactate
○ As methanol is converted to formic acid, pyruvate
molecules are also converted to lactate molecules
○ Lactic acid can also cause the blood to be acidic
● Aside from forming formic acid, you can also form lactic
acid
Metabolic Acidosis
● CLINICAL EFFECTS
○ Kussmaul breathing, dyspnea
○ Headache, nausea, vomiting, confusion, stupor,
coma
○ Decreased myocardial contractility and response
to catecholamine; peripheral vasodilation with
central venoconstriction predisposing to
pulmonary edema; arrhythmias
Butyl Alcohol
● Utilized in plant and animal tissue processing
● Slow dehydrating agent (less miscible with water and
paraffin)
○ Extended hours compared to ethanol
● Produce less tissue shrinkage and hardening than ethyl
alcohol
● Recommended for tissues that do not require rapid
processing
● Flammable, offensive odor and eye irritant (BUTA-nol)
● Since butyl alcohol cannot replace ethanol as dehydrating
agent in the laboratory, it is seldomly used and most of the
time it is used to dehydrate plant and animal tissues for
experiments in botany or zoology or in some researches
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 ● Why can't it replace ethanol?
DEHYDRATION ○ Highly flammable and requires considerable care
● Initial concentration of the alcohol to be used depends on in handling
the nature of the tissue to be process ○ Highly volatile
○ Because if you have delicate and small tissues, ○ Rapid but penetrates tissue poorly
you need to start with low concentrations of ○ Tissues placed in acetone in a prolonged period
alcohol, and the intervals between the increasing of time trends to shrink and to become brittle
concentrations of alcohol should be short ■ Remember: prolonged immersion of
● Smaller and more delicate tissues require lower tissues in ethanol can cause significant
concentration and shorter intervals between change of hardening
succeeding ascending grades of alcohol
○ Prolonged exposure to lower concentrations of
alcohol will macerate or tear the tissue
● A temperature of 37C hestens dehydration and can be
used for urgent biopsy especially for FRAGMENTARY
BIOPSIES
○ If there is a need to shorten the processing time,
you can set the dehydration process at 37 C
○ Fragmentary biopsy
■ Ex. biopsy of the prostate.
● To ensure completeness of dehydration
○ Placed anhydrous copper sulfate with filter paper
at the bottom of the container
○ Anhydrous copper sulfate (white color) turns blue
when the alcohol gets diluted, indicates that the
alcohol has reached the reliability threshold and - Fragmentation due to prolonged immersion of tissue to
need to be changes to ensure the proper acetone
dehydration of the tissues ACETONE IS MORE MISCIBLE IN RESINS THAT ETHANOL
Wrap the white anhydrous copper sulfate in a filter paper Main advantage: no need to heat the chemical to melt it; less chance
↓ of burning
Place the filter paper at the bottom of the container of the absolute
alcohol
↓
Absolute alcohol will remove water molecules from the tissue
↓
The removed water molecules will now be absorbed by the
anhydrous copper sulfate

● The anhydrous copper sulfate somehow speeds up the

removal of water from the tissue
● Take note: once the anhydrous copper sulfate will be
saturated with water, the powder will turn its color from Audio correction “was dehydrated in acetone than in ethanol”
white to blue. So upon checking, if you see that the powder
has already turned blue, this will indicate that the powder is
saturated with water. There is a probability that the absolute
alcohol is no longer 100% alcohol. Thus, there is a need for
you to replace it with a new chemical to ensure complete
removal of water molecules.

ANHYDROUS COPPER SULFATE IS COLORED WHITE BUT IF

EXPOSED WITH WATER, IT TURNS BLUE

Acetone
● Clear colorless fluid that mixes well with water similar to
ethanol
● Cheap rapid acting dehydrating agent (1-2hrs)
● Recommended for urgent biopsies
DIOXANE
● Also known as diethylene dioxide
● Readily miscible with water, alcohol, and paraffin wax
○ No need for clearing because it mixes well with
paraffin wax
○ This reduces time of process
● Produce less tissue shrinkage
○ because tissue was exposed to lesser chemicals
● Tissue can be left immersed for a period of time without
affecting its properties
○ Without significant hardening, tissue shrinkage,
and distortion
● Readily miscible with water, alcohol and paraffin wax
● Readily miscible with water and alcohol (can remove water
from tissues) = dehydration
● Readily miscible with paraffin wax (Impregnation)
● Using dioxane, clearing step can be by passed
THIS IS ONE OF THE METHODS USED IN THE USE OF
DIOXANE. CLEARING IS NOT ANYMORE RECOMMENDED IF
DIOXANE IS USED AS THE DEHYDRATING AGENT
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The following is an example of a time for dehydration with Dioxane
(Graupner’s method)
1st Pure Dioxane Solution 1hr
2nd Pure Dioxane Solution 1hr
3rd Pure Dioxane Solution 2hrs
1st Paraffin Wax 15mins
2nd Paraffin Wax 45mins
3rd Paraffin Wax 2hrs
● Expensive
● Toxic to humans
○ Short exposure - irritation of the eyes, nose and
throat
○ Exposure to large amounts can cause kidney and
liver damage
○ Dioxane = Die!
● Laboratory room should be properly ventilated and frequent
washing of working area is recommended
● Dioxane, if exposed to oxygen in the air can form its
peroxide forms.These peroxide forms are highly explosive
● Since there is not way to test for the presence of this
peroxide forms, recycling of dioxane is greatly prohibited
○ After using the the dioxane for the dehydration
step it's important to dispose it immediately
○ Do not reuse it for another batch of tissue
■ Because everytime we try to open the
container and place the tissue in the
dioxane, we’re allowing air to go into the
container and air brings oxygen and
there's a chance that the oxygen will
react with dioxane forming peroxide and
the lab will explode.
Note
● Tissues which have been treated with chromate fixative
such as Regaud’s fluid should be thoroughly washed in
running tap water prior to treatment with dioxane.
○ Chromate fixatives have sub oxides or oxides in
their chemical composition.
○ If they are not removed, before you immerse the
tissue in dioxane, the oxygen or the oxygen in the
chromate fixatives will react with dioxane and they
will form peroxides.
○ There's a chance that there will be an explosion.
CELLOSOLVE
● Also known as ethylene glycol monoethyl ether
● Advantage: Rapid dehydrating agent
○ It can shorten the processing time and the results
can be released in just few days
● Tissue can be stored in cellosolve for months without tissue
distortion
○ Solve na solve!!!
○ A promising dehydrating agent.
● Disadvantage: Highly flammable L
● Toxic to humans
○ Irritant to the skin, nose, and throat
■ Leading to contact dermatitis
○ May use impotence, kidney damage, toxic effects
to fetus
■ Because cellosolve is known to be toxic
to the cells of the testes.
● Testes- where sperm cells are
produced.
● Exposed male medtechs will
develop oligospermia
(decreased in sperm cell
count) ot azoospermia (total
absence of sperm cells in the
semen)
○ toxic to the bone marrow and testicles. Workers
exposed to high levels are at risk for
granulocytopenia, anemia, oligospermia, and
azoospermia.
○ has been shown to be a teratogen in animal
studies, and is a possible human Teratogen
■ Can cause deformities and mutation in
developing fetuses.
Picture ↑: contact dermatitis secondary to chemical exposure
(ex.cellosolve)
TETRAHYDROFURAN
● Acts as both a dehydrating and a clearing agent similar to
dioxane
● Causes less tissue hardening shrinkage compared to ethyl
alcohol
● Offers good staining results (doesn’t dissolve dyes)
distortions
● As observed, tissues are easy to cut with fewer
● We can actually skip clearing if the dehydrating agent that
we used is Tetrahydrofuran
● Why is it not used as a replacement for ethyl alcohol?
● Exposure to concentrated THF in the workplace has
caused dermatitis and exposure to high levels from
inhalation has been found to be irritating to the eyes, nose
and throat.
● It can cause conjunctival irritation, nausea, headache and
dizziness
● Produces offensive odor
● Requires well ventilated room
Short Quiz
● Can cause oligospermia and azoospermia
○ cellosolve
● Clearing step can be skipped
○ Saman daw be? xD tetrahydrofuran bitaw xD
● Can form peroxides which are highly explosive.
○ dioxane
● Tissues can be immersed in months without tissue
distortion
○ Solve na solve, cellosolve hehe :))
● Prolonged immersion can lead to tissue shrinkage and
increased brittleness
○ acetone
● • Highly flammable and volatile
○ acetone
STAY HYDRATED MADS!! Gudlak sa exam :)))