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Decalcification is the process of removal of
calcium from decalcified tissue and making
suitable for section cutting.
In presence of calcium salts makes the tissue
hard and brittle, which will cause difficulty in
section cutting and damage to the microtome
knife.
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Selection of tissue
Fixation
Decalcification
Detection of end point
Neutralization
Washing
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Thin sizes of bone and other calcified hard
tissues are obtained by using fine toothed
forceps or hack saw .Have to take thickness
of tissue at 4-5 mm.
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Adequate fixation should be needed before
decalcification otherwise tissue will be
damaged in acid decalcification.
Bony tissue is fixed in 10% buffered neutral
formalin for 2-4 days,
For bone marrow zenkers formalin is used.
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Different steps of decalcification are
By using dilute mineral acid
By using ion exchange resins
By chelating agent
Electrophoretic decalcification
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Complete removal of calcium
Minimal tissue damage
Shouldn't interfere the staining reaction
Speed of decalcification
The factors which influence the speed of
decalcification are
Heat
Strength of acid
Agitation
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The acid which is used for decalcification should be simple
solution or mixed with other reagents especially with
fixative or buffered solution.
Different type of decalcifying fluid
Gooding and stewarts fluid
Formic acid(90%)-100ml
Formalin-50ml
Distilled water-850ml
By using formic acid gives a good routine decalcifying
fluid it will give reasonable speed and minimum tissue
damage.
Formaldehyde gives protection to the tissue from acids.
Decalcification by using this solution with in 2-4 days
depends on the thickness and degree of decalcification
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Conc. HCL-15ml
Nacl-175gm
Distilled water-up to 1lt
0.5 % HCl should be added daily till
decalcification is complete.This is a
moderately rapid decalcification.
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Absolute alcohol-73ml
Chloroform-10ml
Acetic acid-3ml
HCl-4ml
Distilled water-10ml
More amount of fluid is needed that is 4050 times the volume of tissue
After decalcification the tissue is directly
transfer to several changes of absolute
alcohol till the acid is removed from the
tissue.
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PH-4.5
7% citric acid monohydrate-5ml
7.54% anhydrous ammonium citrate-95ml
1% zinc sulphate-2ml
Chloroform-2 drops
calcium ions are soluble at PH 4.5
This is slower in action but there is no
damage to the tissue.
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Nitric acid recause the formation of yellow dis
coloration to the tissue and it will interfere
with subsequent staining reaction
The formation of yellow dis coloration can be
prevented by adding 1% urea to pure nitric
acid. But it is having only a temporary effect
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Concentrated HNO3-5-10ml
Distilled water-up to 100ml
It is a good protein decalcifying fluid
Rapid in action but it will cause damage to
the tissue
It will give brilliant staining reaction
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Formalin-5ml
Conc. HNO3-7.5ml
Distilled water-up to 100ml
Formalin prevents the softening effect of
nitric acid on the cell
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Conc. HNO3-10 ml
Phloroglucin-1gm
When bubbling stops at 100 ml of 10%
nitric acid to this solution
Phloroglucin protect the tissue from
softening and gives brilliant staining effect
10% HNO3-40ml
Absolute alcohol-30ml
0.5% chromic acid-30ml
It’s very slow in action for bones. But
excellent for small deposits of calcium
It will cause little hard to the tissue
The end point detection is by x-ray
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It is use to remove calcium ions from the fluid that will
make more rapid rate of solubility of calcium from the
tissue and time taken for decalcification can be reduced
The resins commonly used as ammonium forms of
suphonated poly styrene resins
It’s layered on the bottom of the container to a depth of a
rod 1cm and the specimen is allowed to rest on it
The volume of fluid will be 20-30 times the bulk of the
specimen
Formic acid containing decalcifying fluid will be better
results
After use with resins the tissue must be washed twice in
diluted HCL and followed by washing in running tap water
for 3 times
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These are organic compounds having
capacity to bind with calcium metals
Tissue decalcified by this method showing
minimum of artifacts and good staining
results
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The fixative used is 10% neutral formal saline
After fixation the tissue is transffered to 50
times its bulk of 55% sequestrine buffer of ph
7.4 is prepared in phosphate buffer
The fluid is changed daily for determination
of end point
After decalcification , the tissue is transffered
to 70% alcohol for dehydration
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HILLEMANN’S AND LEE FLUID
EDTA disodium salts – 5.5 gm
Distilled water – 90ml
Formalin – 10ml
NEUTRAL EDTA
It is a cloudy solution it can be neutralised by
adding 2.5 gm of NaOH
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The tissue is placed in electrophoretic tank
containing 2 electrodes and electrolyte
solutions
Equal parts of 8% HCL + 10% Formic acid is
used as an electrolyte
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Tissue should be exposed for longer time in
decalcifying fluid in which it will cause
damage to the tissue
So the end point of decalcification should be
determined to prevent tissue damage and to
ensure the complete removal of calcium
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1) PHYSICAL METHOD
It is a crude method consists of probing the
tissue with a needle and cutting using a
scalpel or should check the flexibility of the
tissue
2) CHEMICAL METHOD
5 ml decalcifying fluid is utilized by strong
ammonia then add 5ml of ammonium oxalate
solution
3) RADIO GRAPHIC METHOD
X-ray
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After decalcification the tissue should be
neutralized with treating with alkali
overnight.
5% Lithium carbonate or NaSO4 can be used
for neutralization
Failure to do the neutralization property that
will cause the swelling of the tissue
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Washing is necessary for removal of alkali
otherwise that will interfere with the staining
reaction
Washing can be done overnight in water or
70% alcohol for 3-5 hrs