2. 2
Contents……………….
• - Introduction
• -Definition
• - Properties of ideal Decalcification agents
• - Methods of Decalcification
• - principle
• -Types of decalcifying agents
• - Compositions, Action, Advantages
• -procedure of Decalcification
3. 3
INTRODUCTION
• Decalcification is necessary in order to
facilitate smooth cutting of bone and other
calcified tissue during the preparation of
sections.
• Calcium is generally present in bones, teeth
and tissues removed from glands such as
tubercular and lymphatic.
• The calcified hard tissue is cut into small
pieces.
• The tissue should be thoroughly washed, to
remove excess of fixative before the
specimen is subjected to decalcification.
5. Criteria of a good decalcifying
Agent
1. Complete removal of calcium
2. Absence of damage to tissue cells or
fibres
3. Non impairment of subsequent
staining technique
4. Reasonable speed of decalcification
6. FACTORS AFFECTING THE
RATE OF
DECALCIFICATION
• 1. Concentration of decalcifying
agent
• 2. Temperature
• 3. Agitation
• 4. Suspension
8
7. FACTORS AFFECTING THE RATE
OF DECALCIFICATION
Concentration of decalcifying agent
• Large volume of the fluid compared with the volume of tissue- 20 to 1
is recommended to avoid total depletion of the acid or chelator by
their reaction with calcium.
• Fluid should be changed several times during the decalcification
process
Temperature
• Increased temperature accelerates decalcification but also
increases
the damaging effects of acids on tissue. 18º C -30º C is
acceptable.
8. Agitatio
n
by
influencin
g
Gentle agitation mayincrease the rate slightly
fluid exchange within as well as around
tissues.
Suspension
Fresh decalcifier should have ready access to all surfaces
of the specimen. enhance diffusion and penetration into the
specimen and facilitate solution, ionization and removal of
calcium.
9. METHODS OF
DECALCIFICATION
1. Acids
2. Ion exchange resins
3. Electrical ionization
4. Histochemical
method
s
Buffer mixtures
Chelating agents
5. Surface decalcification
11. 11
PRINCIPLE
• The acid present in the decalcifying fluid
removes the calcium salt present in the
hard tissue and makes the tissue soft
enough for sectioning.
14. 14
Strongaci
ds
• It Is An InorganicAcid
• Eg: Nitric Acid, HydrochloricAcid
• Recommended Concentration - 5-10%
• They Decalcify Rapidly By Dissolving
Calciu
m
17. 17
1.Fix the selected block of bone for 2-3 days in
buffered neutral formalin.
2.Place a mixture of 95ml distilled water and
5ml of nitric acid.
3.Change nitric acid solutions daily until bubbles
cease to evolve from the tissues(1-3
days,depending on the size and consistency of
the bone block)
4.Wash in 3 changes of 90% alcohol.
5.Dehydrate,clear in xylene or benzene and
embed in paraffin
18. •Formation of nitrous acid checked temporarily
by addition of 0.1% urea to the conc nitric acid
•It’s the fastest decalcifier, but end point must
be carefully watched .
•Yellow discolouration owing to formation of
nitrous acid, this accelerates decalcification but
also stains and damage tissues
18
19. Rapid in action
Gives better nuclear staining
Causes very little hydrolysis
For Needle & Small Biopsy Specimens To
Permit Rapid Diagnosis .
19
Tissue left for long time causes damage to
tissue
Urea is added to remove yellow color of
tissue
21. Weak, organic acids
e.g. formic, acetic, picric.
•Acetic & picric acid cause tissue swelling & are not used
alone as primary decalcifiers but are found as components
of Carnoy’s & Bouin’s fixatives
21
22. •Formicacidisthe only weak acid used
extensively as a decalcifier
•Formic acid solutions are either
• aqueous (5-10%)
• buffered or combined with formalin.
22
23. 23
The formalin/10% formic acid mixture simultaneously fixes &
decalcifies.
• Recommended for –
• Very small bone pieces
• Jamshidi needle biopsies.
• Formic acid gentle & slower than Hcl or nitric acid
• suitable for most routine surgical
specimens, particularly for immuno
histochemistry.
• Decalcification usually complete within 2-7days.
24. Xa)AQUEOUS FORMICACID
1.Well fixed 2-5mm thick blocks are
placed in
– concentrated formic acid
– Distilled water
– 40% formaldehyde
(optional)
5-
25ml
100ml
5 ml
2.Change daily until decalcification is complete.
( 1-7 days for an average blocks depending on concentration of acid.)
3.Replace fluid with 5% sodium sulfate overnight
4.Wash 12 -24 hrs in running tap water.
5.Dehydrate in graded alcohols ,clear in chloroform or toluene and
embed in wax
24
25. DECALCIFICATION PROCEDURE
• Quantity of decal soln >20 vol.of
specimen.
• Wash the decalcified specimen
for 24-48 hrs –to remove the
decal soln.
25
26. 26
PROCEDURE
• 1. Suspend the sliced tissue in the
decalcifying solution by means of gauze
bag, tied with a string.
• 2.Stir the fluid occasionally and change
the fluid daily till calcium is completely
removed from the tissue section.
• 3.It is tested asfollows…….
27. 27
• - take about 5.0 ml of decalcifyingsolution in a
test tube
• - Add conc. Ammonia solution drop by drop
until it becomes alkaline ( test by litmus
paper)
• - If the solution becomes turbid,
continue decalcification ( since it
contains calcium)
• - if it is clear add 0.5 ml saturated ammonium
oxalate solution
• - If calcium is present the solution will become
turbid, then continue decalcification.
• If there is no turbidity , the specimen is free of
calcium and ready for further processing.
28. 28
• 4. Wash the decalcified specimen in
running tap water for 24-48 hours.
• 5.The decalcifying solution should be
completely removed before dehydration
and embedding.
