DECALCIFICATION AND PREPARATION OF GROUND SECTION OF TEETH /certified fixed orthodontic courses by Indian dental academy

INDIAN DENTAL ACADEMY
Leader in continuing Dental Education
DECALCIFICATION AND
PREPARATION OF GROUND SECTION
OF TEETH
www.indiandentalacademy.com

 Decalcification is a process by which
inorganic calcium is removed from organic
collagen matrix after fixation is done but
before dehydration during the preparation of
routine histological sections.
 The calcium present in such tissue not only
hamper section –cutting but may cause
damage to the edge of microtome knife and
hence to be removed.

Choice Of Decalcifier -Depends on
 1)Urgency of the case.
 2)Degree of mineralization.
 3)Extent of investigation.
 4)Staining technique required.

CRITERIA OF A SUITABLE DECALCIFYING
AGENT-
 1)It should be capable of complete removal of
calcium from a tissue.
 2)It should not cause any damage to tissue
cells and fibres.
 3)It should not cause any kind of impairment
to subsequent staining procedure.
 4)It should have a reasonable speed for the
removal of calcium salts from within the tissuewww.indiandentalacademy.com

METHODS OF DECALCIFICATION-There are
three methods-
1)CHEMICAL METHOD-
a) Simple aqueous solution of decalcifying
agent is used.
b) The amount used is 30-40 times of the
tissue sample size,taken in a glass
container.
c) It is kept in a water – bath at room
temperature , preferably 25 degree celsius.www.indiandentalacademy.com

 d)Intermittent shaking of the tissue in the
decalcifying solution is done for a specified
time,depending on the type of agent used.
 e)The sample is tested for ‘end-point’of
decalcification process.
2)ION-EXCHANGE RESIN METHOD-
 a)An ion –exchange resin is used.
 b)Ammonium form of sulphonated polysterene
resin is layered on the bottom of a container
to a depth of half an inch.

 c)The specimen to be decalcified is allowed to
rest on the resin.
 d)A decalcifying agent ,about 10 times the
volume of the specimen is used.
 e)The resin helps to remove the Ca from the
decalcifying agent,thus ensuring more rapid
removal of Ca from the tissue.
 f)The resin can be regenarated after use by
washing with dilute (N/10) HCL acid followed
by washes with distilled water.

3)ELECTROPHORETIC METHOD-
 a)It is a slower procedure but improves the
quality of staining.
 b)In this method a cathode and a anode is
used.
 c) Ca ions being positively charged are
attracted towards negative electrode i.e
cathode.
 d)The electrolyte used is a mixture of equal
parts of 8%HCL and 10% formic acid.
 e)The electrolyte is taken in a glass container.
 f)A perforated perplex sheet is placed

 g)Coiled platinium wire or stainless rod is
used as anode and brass plate or carbon rod
as cathode.
 h)The sample tissue to be decalcified is
placed in the electrolyte near anode.
 i)An electric current of 12 volt is passed
through the electrolyte.
 j)The HCL acid ionizes as H+and CL- and
HCOOH ionizes as H+ and COOH- ions.

 k)The Ca ions in the tissue sample interact
with COOH of formic acid and CL of HCL and
form Ca(COOH)2 and Ca(CL)2 respectively.
 l)Ca(COOH) and Ca(CL)2 splits as Ca++
+2COOH and Ca++ +2CL- respectively.
 j)The Ca ions being positive are repelled by
the anode and hence attracted by the
cathode.

DECALCIFYING AGENTS-Two major types-
 1)Acid reagents and
 2)Chelating agents.
Acid reagents are of two types-
 1)Strong or inorganic acid (Examples are HCL
acid and HNO3 acid).
 2)Weak or organic acids.

Strong or inorganic acid
 They are used as simple aqueous solution in
5-10% concentration.
 They decalcify rapidly but cause tissue
swelling and can damage tissue stability,if
used for longer than 24-48 hours.
 Old acids are more damaging and should be
replaced with fresh stock.
 They are more damaging to tissue antigens
and enzymes may be totally lost.

 Strong acids permit rapid diagnosis within 24
hours.
Weak acids are HCOOH or formic
acids,CCL3COOH or trichloroacetic acid &
picric acid.
 Formic acid may be used as aqueous solution
(5-10%) or combined with formalin.
 Salts like sodium formate or sodium citrate
are added to formic acid solution to make acidwww.indiandentalacademy.com

 Decalcification with formic acid buffer is
usually completed in 1-10 days depending on
size ,type of tissue and acid concentration.
 5%aqueous solution of trichloroacetic acid
needs 12-24 hours to decalcify small tissues.

CHELATING AGENT-EDTA is used.
 It is ethyldiamine tetra acetic acid.
 A solution containing EDTA 10 gm and
distilled water 70 cc having Ph 7 requires 4-40
days to decalcify a tissue depending on size.
 First described by Hileman &Lee in 1963.
 The solution needs to be renewed every
week.
 This agent shows minimum artefact.

Numerous formulas for decalcifying fluids
commonly used are-
 Formic acid-Formic acid (90%) -100ml &
distilled water 900ml.
 Gooding & Stewart fluid (1932)-Formic
acid(90%) 100ml +Formalin 50 ml+ Distilled
water 850 ml.
 Both the above solutions are suitable for
routine use , giving reasonable speed with a
minimum damage to tissue.

 Nitric acid decalcifying fluid contains 80 ml
nitric acid & distilled water 900 ml.
 Its disadvantage is development of yellow
colour which interfere with subsequent
staining.
 This may be avoided by use of 0.1% urea.
 It decalcifies rapidly &a little damage is done
to the tissue if removed as soon as
decalcification is complete.

Factors influencing the rate of decalcification-
 1)Concentration of the decalcifying agent.
 2)Working temperature.
 3)Agitation.
 4)Suspension.
 More concentrated the solution the less is the
time required for decalcification, but it is
harmful to the tissue.

 If buffer or alcohol are added to the agent
then the tissue will be protected but the rate of
decalcification will be slowed down.
 Increased temperature accelerates
decalcification but at the same time it also has
a damaging effect on the tissue.
 The standard temperature is 21 degree
celsius. Room temperature in between 18-30
degree celsius is also acceptable.

 In agitation a tissue processor motor rotating
at the rate of one revolution per minute is
used.
 Doing so reduces the decalcification time from
5-1day.
 Tissue component does not remain intact if
agitation is done at high speed.
 Gentle fluid agitation is achieved by low speed
rotation,rocking,stirring or bubbling air into the
solution.

 Suspension is the procedure by which
decalcifying fluid is made contact with all the
surfaces of the tissue specimen.
 The tissue sample can be suspended in the
fluid with a thread or placed inside cloth bags
tied with threads.

KNOWING DECALCIFICATION ‘END POINT’-
1)Chemical test-In this method after the tissue
has been in the decalcifying fluid for 6-12
hours,5ml of the fluid is taken out from the
bottom of the container.
 5ml each of 5%ammonium hydroxide and 5%
ammonium oxalate is added.
 The two solutions after mixed is allowed to
stand for 15- 20 minutes.
 Appearance of turbidity indicates that the
specimen is not thoroughly decalcified.

 Absence of turbidity indicates complete
decalcification and the tissue should be
removed immediately.
2)Bubble test –Acid reacts with Ca-carbonate to
produce a layer of bubbles on the surface of
the specimen.
 Tiny bubbles indicate less calcium.
 3)Radiography-This is the most sensitive test.

 If not decalcified totally ,areas of
mineralization can be easily identified.
 This method is not applicable when the tissue
is fixed with mercuric chloride as it is opaque
to x-ray.
 Complete decalcification can also be tested
by piercing the hard tissue with a sharp &fine
needle.If penetrates easily ,the tissue is
completely decalcified.

HAZZARDS OF NOT CHANGING TNE
DECALCIFYING AGENT-
 Artefacts can be produced in decalcified tooth
section due to recalcification.
 As the Ca removed from calcified structure
has little opportunity to escape ,it may
precipitate out in the pulp giving rise to
artefactual star shaped crystal aggregates.

DECALCIFICATION OF TEETH-
The objective of decalcification of teeth is to
make the tissue soft.
 During the process enamel is almost lost due
to its high mineral content.
 Before decalcification the teeth should be
fixed whole in NBF.
 Adult teeth require 4 days fixation and for
young teeth where the base of pulp cavity is
more open 24 hour fixation may be adequate.

 The decalcifying agent most commonly used
is 5% Nitric acid or 10% formic acid.
 The fixed tooth is kept in the above
decalcifying agent for 8-10 days changing the
solution daily.
 Brain (1966) used 4M Sodium acetate –HCL
buffer solution at Ph 3.5 for 12 weeks for
decalcification of a tooth.

 Smith (1962) recommended 5%
trichloroacetic acid as a decalcifier.
 Some workers prefer to decalcify with EDTA
or buffered formic acid solution.
 Radiography is regarded ideal for following
the progress and endpoint testing of
decalcification.
 For removal of acid ,the specimen is washed
in running for at least 24 hours after complete
decalcification.

 After that the specimen is taken through
routine procedure like paraffin ,plastic and
celloidin processing,sectioning and adhesion
techniques and staining.

PREPARATION OF GROUND SECTION OF
TOOTH-
Ground section of teeth are essential for the
study of enamel & to some extent dentin &
cementum.
 Enamel has very high content of
calcium,about 96% of its weight.
 Hence it gets completely destroyed during
decalcification.

 Apparatus with which undecalcified teeth can
be cut and polished have been designed
primarily for cutting ,grinding &polishing
geological specimens.
 In C.F.A.Culling’s laboratory teeth are
embeded in a cold curing methacrylate.
 Sections about 100 micro meter thick cut on a
microslice 2.

 Cutting is effected by a diamond -edged disc
,which may be of annular or peripheral
configuration.
 Annular cutting discs are more accurate &
less prone to distortion.
 The cutting procedure is cooled with a
continuous drip feed of water or light oil.

LABORATORY PREPARATION OF
GROUND SECTION OF TOOTH-To obtain a
thin ground section of a tooth, it is cut
logitudinally in a mesiodistal plane.
 A coarse abrasive carborandum disc is
attached to a dental micromotor.
 The tooth is held securely in the fingers while
water is sprayed on the tooth.

 The buccal surface is applied firmly to the
rapidly rotating carborandum disc.
 A fine abbrasive lathe wheel is then used to
ground the tooth.
 To direct the other side outward ,a 13 mm
adhesive tape is wrapped round a wooden
block.
 The ground surface of the tooth is wiped dry
and pressed onto the adhesive tape on a side
of the wooden block so that it sticks firmly.

 The lingual surface of the tooth is then applied
to the coarse adhesive lathe wheel and
ground to 0.5 mm.
 With a fine abbrasive lathe wheel the section
is further ground to a thickness of 50- 70
microns.
 Ether is used to remove the adhesive tape
from the ground section.

 The section is further ground to a thickness of
30-50 microns on a fine and smooth flat
carborundum stone slab.
 Ground can also be prepared on grinding
stone slabs of various grades using pumice
and water paste.
 The section is then dried for several minutes.
 Care should be taken that small particles of
abrasive does not get embeded into the
section.

 The section is lifted with a camel’s hair brush.
 It is then placed on a drop of mounting
medium placed on a glass slide.
 Another drop of mounting medium is put on
the section and covered with a cover glass.
 The slide is ready for examination under
microscope.

DECALCIFICATION AND PREPARATION OF GROUND SECTION OF TEETH /certified fixed orthodontic courses by Indian dental academy

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