24. DECALCIFICATION LAMBERT

DECALCIFICATION OF BIOPSY TISSUE
BY
Dr. Archana S.
B.D.S., M.D.S. (Oral Pathology)
Consulting Oral & Maxillofacial Pathologist
Ex. Post Graduate Resident,
Dept. of Oral Pathology & Microbiology,
JSS Dental College & Hospital,
JSS University,
Mysuru, Karnataka, India
Dr. Usha Hegde
B.D.S., M.D.S. (Oral Pathology)
Professor & Head,
JSS University,
Dr. Bhuvan Nagpal
B.D.S. (Hons.), M.D.S. (Oral Pathology)
(Gold Medalist)
Consulting Oral & Maxillofacial Pathologist
Ex. Post Graduate Resident,
JSS University,

Sl.No CONTENTS Page No.
1. INTRODUCTION 3
2. TECHNIQUE OF DECALCIFICATION 5
3. DECALCIFYING AGENTS 11
4. FACTORS INFLUENCING DECALCIFICATION 33
5. METHODS OF DECALCIFICATION 38
6.
HISTOCHEMICAL METHODS FOR
DECALCIFICATION
47
7. END POINT DETERMINATION 51
8. ARTIFACTS IN DECALCIFICATION 57
9. CONCLUSION 61
10. REFERENCES 63

INTRODUCTION
Teeth and bone belong to the category of hardest tissues, which is denser and
chemically more inert than other body tissues. Because of large amounts of
inorganic components that is calcium and phosphorus, the biological apatite is very
hard to prepare for microscopic examinations.1
Human teeth, as well as alveolar bone, must be decalcified during processing for
histologic analysis because of its structure. Rapid fixation of all dental elements is
difficult to obtain because penetration of the fixative agent through such structures
as enamel, dentin and bone is a slow process. In such cases, the tissues in the center
of the specimen may undergo some alterations before fixation is completed. The
most seriously affected tissue is perhaps the pulp.1, 2
The goal of decalcification is to remove calcium salts from the mineralized tissues
and preparing them for further sectioning of the histologic specimen. Any acid,
even if properly buffered, affects tissue stability. These effects depend on the
solution’s acidity and duration of the decalcification process.3
In addition, the faster
the action of the decalcifying agent, the greater the damage to the tissue. The rapidity
of decalcification may also lead to untoward effects to the staining technique
performed subsequently. There are various factors influencing the speed of
decalcification such as decalcifying solution concentration, temperature, stirring and
tissue suspension.1,3
Decalcification is performed by chemical solutions, which employ acids (acids may
be divided into strong and weak acids or chelates). It is thought to be emphasized
that fixative agents that contain acid in their composition, such as formalin (that
contains formic acid), may also be able to act as decalcifying agents if the acid
component is not neutralized.2
Decalcification is commonly employed in most histopathology laboratories for the
microscopic examination of bone and other calcified tissues. Plastic processing
without decalcification may produce superior results in terms of eliminating

shrinkage and for demonstrating osteoid versus mineralized matrix but may give
poor cytological detail and is a much longer process2
. The diagnosis of non-
metabolic diseases of bone such as infections and tumors requires good cellular
morphology and a quick result to allow rapid therapeutic intervention for optimal
patient care.4
Most unsatisfactory results with decalcification can be attributed to overexposure to
the agent used and due to inadequate control procedures. Many alternative
decalcification regimen have been proposed, but most of them have atleast a few
unsatisfactory characteristics. In an attempt to reduce the commonly encountered
artifacts of tissue shrinkage and adverse staining results obtained with rapid
decalcification using strong mineral acids such as nitric acid, a rapid method of
decalcification was devised which gave excellent and reproducible results.2,3
The
procedure for decalcification and the successful monitoring of the process is
discussed and some popular options for the choice of reagents are provided.
DECALCIFICATION:
In order to obtain satisfactory sections of bone, inorganic calcium must be
removed from the organic collagen matrix, calcified cartilage, and surrounding
tissues. This is called decalcification.2

TECHNIQUE OF DECALCIFICATION
The technique of decalcification is divided into following stages:
1. Selection of tissue
2. Fixation
3. Decalcification
4. Neutralization of acid
5. Tissue processing & Staining
The above scheme sets out a general plan of work, but it should not be taken
as rigid schedule. On occasion, fixation of a gross specimen will precede
selection of a piece for decalcification, and there are certain fluids which have
a fixing and decalcifying action.1,2
1) SELECTION OF TISSUE:
Thin slices of bone are obtained using a fine -toothed bone saw, or hacksaw.
To ensure adequate fixation and complete removal of the calcium, slices should
not exceed 4-5 mm in thickness.5
Further the cut surfaces should be re-trimmed
to remove the areas damaged by the saw. Thin slices of calcified tissue can
usually be cut with a sharp knife, but when difficulty is encountered a saw
should be used to avoid damage to the tissue surrounding the calcified area.
The type and duration of the treatment of such tissues ( for example, chronic
tuberculosis foci, calcified scar tissue) will depend on the degree of
calcification. Tissues containing only small areas should be tested after 2-3
hours in a decalcifying fluid.2,5
2) FIXATION:
In order to protect the cellular and fibrous elements of bone from damage caused by
the acids used as decalcifying agents, it is particularly important to thoroughly fix
these specimens prior to decalcification.1
Poorly-fixed specimens become macerated
during decalcification and stain poorly afterwards. This is very noticeable in areas

containing bone marrow. It is therefore common practice for laboratories to extend
fixation times for bone specimens before commencing decalcification. It is
important to provide ready access for the fixative to penetrate the bone, so skin and
soft tissue should be removed from large specimens if practicable.5
Bone specimens
should be sawed into thin slices as soon as possible to enhance fixation. An adequate
volume of fixative should be used. High-quality fine tooth saws should be used to
prepare bone slices. Coarse saws can cause considerable mechanical damage and
force bone fragments into the soft tissues present in the specimen.6
In case of
tooth, the pulpal fixation is done by trimming the apical end of tooth until the
pulp is exposed and then injecting formalin in the apical area where pulp is
fixed.2,6
Buffered formalin is a satisfactory fixative for bone, but where the preservation of
bone marrow is important some laboratories will use alternatives such as one of
the Zinc formalin mixtures, formol acetic alcohol (Davidson’s fixative), or Bouin
fluid. Bone marrow is best fixed in Zenker formol.7
Some fine preparations of
bone have been produced following immersion in Mullers fluid for upto 3
months, followed by decalcification in 3% formic-acid-formalin.6
It has been shown by Cook and Ezra Cohn(1962) that tissue damage during
acid decalcification is approximately four times greater when the tissue is
unfixed.7
3) DECALCIFICATION:
Pierre de Coubertin proposed the Olympic motto “citius, altius, fortius” which is
Latin stands for faster, higher, and stronger respectively.5
There are different
decalcifying agents such as acids and chelating agents, Gray (1954) lists over
50 different mixtures. Many of these mixtures were developed for special purposes.
One such mixture was used as a fixing and dehydrating agent, Other
mixtures contain reagents, like buffer salts, chromic acid, formalin or ethanol,
intended to counteract the undesirable swelling effects that acids have on

tissues.8
Many popular mixtures used today are from the original formulas
developed many years ago (Evans & Krajian 1930; Kristensen 1948; Clayden
1952). For most practical purposes, today’s laboratories prefer simpler solutions
for routine work; If the bone is totally fixed and then treated with a decalcifier
suitable for removal of mineral, the simple mixtures work as well or better than
more complex mixtures.7, 8
There are three main types of decalcifying agents:
• Those based on strong mineral acids
• Those based on weaker organic acids
• Those composed of chelating agents.1, 2
The criteria of good decalcifying agents are:
• Complete removal of calcium
• Absence of damage to tissue cells or fibres
• Non-impairment of subsequent staining techniques
• Reasonable speed of decalcification.2, 3
4) NEUTRALIZATION OF ACID
• Acids can be removed from tissues or neutralized chemically after
decalcification is complete.
• Chemical neutralization is accomplished by immersing decalcified bone into
either saturated lithium carbonate solution or 5-10% aqueous sodium
bicarbonate solution for several hours. Many laboratories simply rinse the
specimens with running tap water for a period of time.9
• Culling (1974) recommended washing in two changes of 70% alcohol for 12-
18 hours before continuing with dehydration in processing, a way to avoid
contamination of dehydration solvents even though the dehydration process
would remove the acid along with the water.4

• Adequate water rinsing can generally be done in 30 minutes for small samples
and 1-4 hours for larger bones. For cryomicrotomy acid decalcified tissues
must be washed in water and stored in formol saline containing 15% sucrose
or PBS at 40
C before freezing.2
• Tissues decalcified in EDTA solutions should not be placed directly into 70%
alcohol as this causes residual EDTA to precipitate in the alcohol and within
the tissue. The precipitate does not appear to affect tissue staining, since
EDTA is washed out during these procedures, but may be noticeable during
microtomy or storage when a crystalline crust forms on the block surface. A
water rinse after decalcification or over-night storage in formol saline or PBS
should prevent this.10,11
5) TISSUE PROCESSING & STAINING
• Small blocks containing small amounts of bone may be processed routinely.
Larger or dense blocks require special attention. Often bone infiltrated with
routine paraffin for all tissues can be embedded in harder paraffin to give
firmer support to bone during sectioning.
• The processing should be prolonged with extended periods in molten wax
with three changes of wax under vacuum of 2 hours each.16
MICROTOMY:
A substantial microtome and knife contribute greatly to successful microtomy of
bone. A base sledge microtome and wedge-shaped steel or tungsten carbide edged
knife are recommended. The rake of the knife should be less than for conventional
microtomy, even though this may result in some compression. The blocks should
be well iced before cutting. Slightly thicker sections, 6-7 are acceptable when
cutting bone.11
It often helps if tissue is embedded obliquely in the wax. The floating out bath may
need to be hotter than for soft tissues, as bone has a tendency to crinkle when cut.

Sections should be picked up on chrome gelatine coated slides, to reduce the chance
that they may lift from the slide during staining.15
Fig 16: Microtome17
LENDRUMS TECHNIQUE:
It is very useful method treating tissues known to be hard, hard tissue which is
difficulty in cutting is encountered in spite of, or in the absence of, early precautions,
the block should be soaked in Mollifex (obtainable from British drug houses)
overnight & cut in the usual way the next morning. Although the surface will have
a soapy consistency after this treatment sections cut & stain quite well.2
STAINING:
• Acid treated tissue is less susceptible to hematoxylin staining.
• Ehrlich’s hematoxylin is a good nuclear stain in these conditions and has the
added advantage that it stains mucopolysaccharides and thus demonstrates
cement lines (or reversal lines) and cartilage well.
• Gill’s haematoxylin (3 x) is also a useful stain for decalcified bone. Eosin
staining is enhanced by acid decalcification, so that some reduction in staining
time is required.
• General bone structure is well demonstrated by silver reticulin methods and
particularly by picrothionin.17

DECALCIFYING AGENTS
I. STRONG ACIDS:
Strong acids such as hydrochloric or nitric acid at concentrations up to 10%
are the most rapid in action but if used for an excessive time will rapidly cause a
loss of nuclear staining and can macerate tissues.9
It is important that an
appropriate end-point test is used to minimize exposure of the specimens to these
agents. Generally proprietary decalcifiers that are claimed to be rapid in action are
based on strong acids, most commonly hydrochloric acid, and should be used
conservatively with attention to the provided instructions if good results are to be
obtained. For example Surgipath’s Decalcifier II is rapid in action and contains
hydrochloric acid.9,10
1. NITRIC ACID
History:
The first mention of nitric acid is in Pseudo-Geber's De Inventione Veritatis,
wherein it was obtained by calcining a mixture of niter, alum and blue vitriol. It was
again described by Albert the Great in the 13th century and by Ramon Lull, who
prepared it by heating niter and clay and called it "eau forte" (aqua fortis).1, 9
Glauber devised the process still used today by heating niter with strong
sulfuric acid. In 1776 Lavoisier showed that it contained oxygen, and in
1785 Henry Cavendish determined its precise composition and showed that it could
be synthesized by passing a stream of electric sparks through moist air.10
Nitric acid (HNO3), also known as aqua fortis and spirit of niter, is a
highly corrosive mineral acid. The pure compound is colorless, but older samples
tend to acquire a yellow cast due to decomposition into oxides of nitrogen and water.
Most commercially available nitric acid has a concentration of 68%.2
when the
solution contains more than 86% HNO3; it is referred to as fuming nitric acid.
Depending on the amount of nitrogen dioxide present, fuming nitric acid is further

characterized as white fuming nitric acid or red fuming nitric acid, at concentrations
above 95%.9,10
Nitric acid is the primary reagent used for nitration the addition of a nitro group,
typically to an organic molecule. While some results of nitro compounds are
shock- and thermally-sensitive explosives, a few are stable enough to be used in
munitions and demolition, others are still more stable and used as pigments in
inks and dyes. Nitric acid is also commonly used as a strong oxidizing agent.10
• Physical and chemical properties:
Commercially available nitric acid is an azeotrope with water at a concentration of
68% HNO3, which is the ordinary concentrated nitric acid of commerce. This
solution has a boiling temperature of 120.5 °C at 1 atm. Two solid hydrates are
known; the monohydrate (HNO3·H2O) and the trihydrate (HNO3·3H2O). Nitric acid
of commercial interest usually consists of the maximum boiling azeotrope of nitric
acid and water, which is approximately 68% HNO3, (approx. 15 molar). This is
considered concentrated or technical grade, while reagent grades are specified at
70% HNO3. The density of concentrated nitric acid is 1.42 g/mL.9,10
TABLE 1: Properties of nitric acid18
Properties
Molecular formula HNO3
Molar mass 63.01 g mol−1
Appearance Colourless liquid
Density 1.5129 g cm−3
Melting point −42 °C (−44 °F; 231 K)
Boiling point 83 °C (181 °F; 356 K) 68% solution boils
at 121 °C (250 °F; 394 K)
Solubility in water Completely miscible
Acidity (pKa) -1.4
Refractive index(nD) 1.397 (16.5 °C)
Dipole moment 2.17 ± 0.02 D

Fig 1: Structure of nitric acid18
• Laboratory synthesis:
In laboratory, nitric acid can be made by thermal decomposition of copper (II)
nitrate, producing nitrogen dioxide and oxygen gases, which are then passed through
water to give nitric acid.9
2 Cu(NO3)2 2 CuO (s) + 4 NO2 (g) + O2 (g)
An alternate route is by reaction of approximately equal masses of any nitrate salt
such as sodium nitrate with 96% sulfuric acid (H2SO4), and distilling this mixture at
nitric acid's boiling point of 83°C. A nonvolatile residue of the metal sulfate remains
in the distillation vessel. The red fuming nitric acid obtained may be converted to
the white nitric acid.9,10
2 + H2SO4 2 HNO3 +
The dissolved NOx {Generic term for the mono-nitrogen oxides NO and NO2 (nitric
oxide and nitrogen dioxide)}are readily removed using reduced pressure at room
temperature (10–30 min at 200 mmHg or 27 kPa) to give white fuming nitric acid.
This procedure can also be performed under reduced pressure and temperature in
one step in order to produce less nitrogen dioxide gas.10
Dilute nitric acid may be concentrated by distillation up to 68% acid, which is a
maximum boiling azeotrope containing 32% water. In the laboratory, further

concentration involves distillation with either sulfuric acid or magnesium
nitrate which acts as dehydrating agents. Such distillations must be done with all-
glass apparatus at reduced pressure, to prevent decomposition of the acid.
Industrially, highly concentrated nitric acid is produced by dissolving additional
nitrogen dioxide in 68% nitric acid in an absorption tower. Dissolved nitrogen
oxides are either stripped in the case of white fuming nitric acid, or remain in
solution to form red fuming nitric acid. More recently, electrochemical means have
been developed to produce anhydrous acid from concentrated nitric acid feedstock.8
Xanthoproteic test:
Nitric acid reacts with proteins to form yellow nitrated products. This reaction is
known as the xanthoproteic reaction. This test is carried out by adding concentrated
nitric acid to the substance being tested, and then heating the mixture. If proteins
that contain amino acids with aromatic rings are present, the mixture turns yellow.
Upon adding a strong base such as liquid ammonia, the color turns orange. These
color changes are caused by nitrated aromatic rings in the protein. Xanthoproteic
acid is formed when the acid contacts epithelial cells and is indicative of inadequate
safety precautions when handling nitric acid.9
• Safety:
Nitric acid is a corrosive acid and a powerful oxidizing agent. The major hazard
posed by it is chemical burns as it carries out acid hydrolysis with proteins (amide)
and fats (ester)which consequently decomposes living tissue (e.g.skin and flesh).
Concentrated nitric acid stains human skin yellow due to its reaction with
the keratin. These yellow stains turn orange when neutralized. Systemic effects are
unlikely, however, and the substance is not considered a carcinogen or mutagen.10
The standard first aid treatment for acid spills on the skin is, as for other corrosive
agents, irrigation with large quantities of water. Washing is continued for at least ten
to fifteen minutes to cool the tissue surrounding the acid burn and to prevent
secondary damage. Contaminated clothing is removed immediately and the
underlying skin washed thoroughly. Being a strong oxidizing agent, reactions of

nitric acid with compounds such as cyanides, carbides, metallic powders can
be explosive and those with many organic compounds, such as turpentine, are
violent and hypergolic (i.e. self-igniting). Hence, it should be stored away from
bases and organics.9,10
• FLUIDS CONTAINING NITRIC ACID:
Formol nitric acid:
Formula:
• Formalin…………………..5 ml
• Nitric acid (specific gravity 1.41)……………7.5 – 15 ml
• Distilled water ……………to 100 ml.
-In this formula, the formaldehyde partially inhibits the tendency to maceration
by the nitric acid.
-In practice aqueous nitric acid give better cell preservation and staining.
-Discoloration may be prevented by stabilization with urea.2,9
Phloroglucin – Nitric acid
1. Place 10 ml of nitric acid (specific gravity 1.41) in an evaporating dish.
2. Add 1 g of phloroglucin.
3. When bubbling ceases add 100 ml of 10 % nitric acid.
• The use of phloroglucin is said to protect the tissue from maceration, and
allows good subsequent staining.
• But according to some, decalcification here is very rapid, subsequent
staining is very poor, and the method cannot be recommended.2
Aqueous Nitric acid:
Formula
• Nitric acid (stabilized with 0.1% urea)…. 5- 10 ml
• Distilled water………….to 100 ml.

The fluid recommended by Clayden is used as routine decalcifying agent. It
is rapid, causes little damage to tissue if the time of decalcification is carefully
controlled, and allows most staining techniques to be applied.9
Perenyi’s Fluid:
Formula
• 10% nitric acid……………..40 ml
• Absolute alcohol……………30 ml
• 0.5% chromic acid ………..30 ml.
These solutions are kept in stock and mixed freshly when required.
The solution acquires a violet tinge after a short while.
Perenyi’s fluid is slow for decalcifying dense bone.
Is an excellent reagent for small deposits of calcium.
It has little hardening effect on tissue and excellent cytological preparations
are possible after its use.
The chemical test for decalcification cannot be carried out with this fluid : x-
rays should be used.10
Advantage:
Many writers highly recommend 5%, which causes no swelling and acts
powerfully.2
Disadvantages:
• A disadvantage of using nitric acid as a decalcifying fluid is the yellow colour
which develops owing to the formulation of nitrous acid.
• This causes alteration in the speed of decalcification – it gets more rapid as
the colour develops.

• It also causes yellow discoloration of the tissue which subsequently interferes
with staining reactions.
• The yellow colour can be obviated by the addition of 0.1% urea to pure
nitric acid , which should be colourless.
• Even brief exposure to this acid renders unsatisfactory staining of bone
marrow with Giemsa, Maximow's azure ll-eosin or Lillie's azure eosin
formula.2,10
• As the urea has only a temporary effect, further additions should be made
when the acid becomes tinged with yellow(Clayden,1952).1
2. HYDROCHLORIC ACID:
• Etymology:
Hydrochloric acid was known to European alchemists as spirits of salt or
acidumsalis (salt acid). Both names are still used, especially in non English
languages, such as German: Salzsäure, Dutch: Zoutzuur, Northern
Sami: Saltsyra and Polish: kwas solny. Gaseous HCl was called marine acid air.
The old (pre-systematic) name muriatic acid has the same origin (muriatic means
"pertaining to brine or salt"), and this name is still sometimes used. The name
"hydrochloric acid" was coined by the French chemist Joseph Louis Gay-Lussac in
1814.11
• History
Aqua regia, a mixture consisting of hydrochloric acid and nitric acid, prepared by
dissolving salt ammoniac in nitric acid, was described in the works of Pseudo-
Geber, the 13th-century European alchemist.9
Other references suggest that the first
mention of aqua regia is in Byzantine manuscripts dating to the end of the thirteenth
century. Free hydrochloric acid was first formally described in the 16th century
by Libavius, who prepared it by heating salt in clay crucibles. Other authors claim
that pure hydrochloric acid was first discovered by the German benedictine

monk Basil Valentine in the 15th century, by heating common salt and
green vitriol, whereas others claim that there is no clear reference to the preparation
of pure hydrochloric acid until the end of the sixteenth century.10,11
In the seventeenth century, Johann Rudolf Glauber from Karlstadt am Main,
Germany used sodium chloride salt and sulfuric acid for the preparation
of sodium sulfate in the Mannheim process, releasing hydrogen
chloride gas. Joseph Priestley of Leeds, England prepared pure hydrogen chloride
in 1772, and by 1808 Humphry Davy of Penzance, England had proved that the
chemical composition included hydrogen and chlorine.9
During the Industrial Revolution in Europe, demand for alkaline substances
increased. A new industrial process by Nicolas Leblanc (Issoundun, France)
enabled cheap large-scale production of sodium carbonate (soda ash). In
this Leblanc process, common salt is converted to soda ash, using sulfuric acid,
limestone, and coal, releasing hydrogen chloride as a by-product. Until the
British Alkali Act 1863 and similar legislation in other countries, the excess HCl
was vented to air. After the passage of the act, soda ash producers were obliged to
absorb the waste gas in water, producing hydrochloric acid on an industrial scale.11,12
In the twentieth century, the Leblanc process was effectively replaced by the Solvay
process without a hydrochloric acid by-product. Since hydrochloric acid was already
fully settled as an important chemical in numerous applications, the commercial
interest initiated other production methods, some of which are still used today. After
the year 2000, hydrochloric acid is mostly made by absorbing by-product hydrogen
chloride from industrial organic compound production.10
• Physical and Chemical Properties:
Hydrogen chloride (HCl) is a monoprotic acid, which means it
can dissociate (i.e., ionize) only once to give up one H+
ion (a single proton). In
aqueous hydrochloric acid, the H+
joins a water molecule to form a hydronium ion,
H3O+
.

HCl + H2O H3O+
+ Cl−
The other ion formed is Cl−
, the chloride ion. Hydrochloric acid can therefore be
used to prepare salts called chlorides, such as sodium chloride. Hydrochloric acid is
a strong acid, since it is essentially completely dissociated in water.11
Of the six common strong mineral acids in chemistry, hydrochloric acid is the
monoprotic acid least likely to undergo an interfering oxidation-
reduction reaction.10
It is one of the least hazardous strong acids to handle; despite
its acidity, it consists of the non-reactive and non-toxic chloride ion. Intermediate-
strength hydrochloric acid solutions are quite stable upon storage, maintaining their
concentrations over time. These attributes, plus the fact that it is available as a
pure reagent, make hydrochloric acid an excellent acidifying reagent.12
Hydrochloric acid is the preferred acid in titration for determining the amount
of bases. Strong acid titrants give more precise results due to a more distinct
endpoint. Azeotropic or "constant-boiling" hydrochloric acid (roughly 20.2%) can
be used as a primary standard in quantitative analysis, although its exact
concentration depends on the atmospheric pressure when it is prepared.11
Hydrochloric acid is frequently used in chemical analysis to prepare ("digest")
samples for analysis. Concentrated hydrochloric acid dissolves many metals and
forms oxidized metal chlorides and hydrogen gas, and it reacts with basic
compounds such as calcium carbonate or copper (II) oxide, forming the dissolved
chlorides that can be analyzed.12

Fig 2 : Structure of Hydrochloric acid18
Physical properties of hydrochloric acid, such as boiling and melting
points, density, and pH, depend on the concentration or molarity of HCl in the
aqueous solution. They range from those of water at very low concentrations
approaching 0% HCl to values for fuming hydrochloric acid at over 40% HCl.9
• Presence in living organisms:
Gastric acid is one of the main secretions of the stomach. It consists mainly of
hydrochloric acid and acidifies the stomach content to a pH of 1 to 2.11
• Safety:
Concentrated hydrochloric acid (fuming hydrochloric acid) forms acidic mists. Both
the mist and the solution have a corrosive effect on human tissue, with the potential
to damage respiratory organs, eyes, skin, and intestines irreversibly. Upon mixing
hydrochloric acid with common oxidizing chemicals, such as sodium
hypochlorite (bleach, NaClO) or potassium permanganate (KMnO4), the toxic
gas chlorine is produced.11,12
NaClO + 2 HCl H2O + NaCl + Cl2
2 KMnO4 + 16 HCl 2 MnCl2 + 8 H2O + 2 KCl + 5 Cl2

Personal protective equipment such as rubber or PVC gloves, protective eye
goggles, and chemical-resistant clothing and shoes are used to minimize risks when
handling hydrochloric acid. The United States Environmental Protection
Agency rates and regulates hydrochloric acid as a toxic substance.10
FLUIDS CONTAING HYDROCHLORIC ACID:
Jenki’s fluid:
Formula
Absolute alcohol………..73 ml
Distilled water……10 ml
Chloroform……….3 ML
Glacial acetic acid……..3 ml
Hydrochloric acid……….4 ml
*Jenki’s fluid not only decalcifies but also dehydrates. The swelling action of the
hydrochloric acid is counteracted by shrinkage effect of the alcohol.
*Large amounts of this fluid should be used, between 40 & 50 times the bulk of the
tissue. After decalcification the tissue is transferred directly to absolute alcohol in
which it is given several changes to remove the acid.
*Cross- section of human rib is decalcified in 4-6 days.9
Von Ebners fluid:
Formula
• Concentrated hydrochloric acid…..15 ml
• Sodium chloride…..175g
• Distilled water…. To 1,000 ml

* Hydrochloric acid (0.5%) should be added daily until decalcification is complete.
*This fluid is popular in certain parts of Great Britain as a routine decalcifying agent.
It is moderately rapid in action, not quite as good as those obtained with Gooding &
Stewart’s, or Perenyi’s fluid.
*Cross section of human rib (5 mm) is decalcified in 36-72 hours.2,9
Advantages:
Action is rapid, even in dilute solutions. This acid, when used at 370
C, preserves
eosin stains of cytoplasm. When used at 150
to 250
C, satisfactory H & E, Van
Gieson, Masson, and azure eosin stains may be done, if exposure is not prolonged.
To remedy swelling of tissues, chromic acid or alcohol may be added to the solution.
l5% NaCl may also be added to a 3% acid solution to counteract the swelling
action.2,12
Disadvantages:
Causes serious swelling of tissue. At 550
to 600
C loss of calcium salts occurs rapidly,
followed by swelling and hydrolysis of bone matrix, which soon results in complete
digestion. This occurs in as little as 24 hr in 8% hydrochloric acid. Decalcification
at 370
C impairs alum hematoxylin staining and Weigert's iron hematoxylin staining
of nuclei to some extent. Feulgen staining of nuclei is unsuccessful and azure eosin
stains give pink cytoplasm and nuclei.12
II.WEAK ACIDS:
Formic is the only weak acid used extensively as a primary decalcifier. Acetic and
picric acids cause tissue swelling and are not used alone as decalcifiers but are found
as components in Carnoy's and Bouin's fixatives. These fixatives will act as
incidental although weak, decalcifiers and could be used in urgent cases with only
minimal calcification. Formic acid solutions can be aqueous (5-10%), buffered or
combined with formalin.9

The formalin-10% formic acid mixture simultaneously fixes and decalcifies, and is
recommended for very small bone pieces. However, it is still advisable to have
complete fixation before any acid decalcifier is used. Formic acid is gentler and
slower than HC1 or nitric acid, and is suitable for most routine surgical specimens,
particularly when immunohistochemical staining is needed. Formic acid can still
damage tissue, antigens and enzyme histochemical staining, and should be end-point
tested.10
Decalcification is usually complete in 1-10 days, depending on the size, type of bone
and acid concentration. Dense cortical or large bones have been effectively
decalcified with 15% aqueous formic acid and a 4% HCl- 4% formic acid mixture.13
1. FORMIC ACID
• Introduction:
Formic acid (systematically called methanoic acid) is the simplest carboxylic acid.
Its chemical formula is HCOOH or HCO2H. It is an important intermediate
in chemical synthesis and occurs naturally, most notably in ant venom. Its name
comes from the Latin word for ant, Formica, referring to its early isolation by
the distillation of ant bodies. Esters, salts, and the anions derived from formic acid
are referred to as formates.2,13
• History:
Some alchemists and naturalists were aware that ant hills give off an acidic vapor as
early as the 15th century. The first person to describe the isolation of this substance
(by the distillation of large numbers of ants) was the English naturalist John Ray, in
1671. Ants secrete the formic acid for attack and defense purposes. Formic acid was
first synthesized from hydrocyanic acid by the French chemist Joseph Gay-Lussac.
In 1855, another French chemist, Marcellin Berthelot, developed a synthesis from
carbon monoxide that is similar to that used today.10,13

Formic acid was long considered a chemical compound of only minor interest in the
chemical industry. In the late 1960s, however, significant quantities of it became
available as a byproduct of acetic acid production. It now finds increasing use as a
preservative and antibacterial agent in livestock feed.13
• Physical and chemical properties:
Formic acid is a colorless liquid having a highly pungent, penetrating odorant room
temperature. It is miscible with water and most polar organic solvents, and is some
what soluble in hydrocarbons. In hydrocarbons and in the vapor phase, it consists
of hydrogen-bonded dimers rather than individual molecules. Owing to its tendency
to hydrogen-bond, gaseous formic acid does not obey the ideal gas law. Solid formic
acid (two polymorphs) consists of an effectively endless network of hydrogen-
bonded formic acid molecules.2,9
Fig 3: Structure of Formic acid18

Table 2: Properties of Formic acid18
Properties
Molecular formula CH2O2
Appearance colourless fuming liquid
Odour pungent, penetrating
Density 1.220 g/Ml
Melting point 8.4 °C (47.1 °F; 281.5 K)
Boiling point 100.8 °C (213.4 °F; 373.9 K)
Solubility in water Miscible
Solubility miscible with ether, acetone, ethyl acetate, glycerol,
methanol, ethanol
partially soluble in benzene ,toluene.
log P −0.54
Acidity (pKa) 3.77
Refractive
index(nD)
1.3714 (20 °C)
Viscosity 1.57 cP at 268 °C
• Laboratory use:
Formic acid is a source for formyl group for example in the formylation of
methylaniline to N-methylformanilide in toluene. In synthetic organic chemistry,
formic acid is often used as a source of hydride ion. The Eschweiler-Clarke
reaction and the Leuckart-Wallach reaction are examples of this application. It, or
more commonly its azeotrope with triethylamine, is also used as a source of
hydrogen in transfer hydrogenation.13
As mentioned below, formic acid may serve as a convenient source of carbon
monoxide by being readily decomposed by concentrated sulfuric acid.10
CH2O2(l) + H2SO4(l) H2SO4(l) + H2O(l) + CO(g)
• Safety:
Formic acid has low toxicity (hence its use as a food additive), with an LD50 of 1.8
g/kg (oral, mice). The concentrated acid is, however, corrosive to the skin.9

Formic acid is readily metabolized and eliminated by the body. Nonetheless, it has
specific toxic effects; the formic acid and formaldehyde produced as metabolites
of methanol are responsible for the optic nerve damage, causing blindness seen in
methanol poisoning. Some chronic effects of formic acid exposure have been
documented. Some experiments on bacterial species have demonstrated it to be
a mutagen. Chronic exposure to humans may cause kidney damage. Another
possible effect of chronic exposure is development of a skin allergy that manifests
upon re-exposure to the chemical.12
Concentrated formic acid slowly decomposes to carbon monoxide and water,
leading to pressure buildup in the container it is kept in. For this reason, 98% formic
acid is shipped in plastic bottles with self-venting caps.13
• FLUIDS CONTAINING FORMIC ACID:
Aqueous Formic Acid:
Formula
• 90%stock formic acid……………. 5-10 ml
• Distilled water……………to 100 ml
Formic Acid—Formalin (After Gooding & Stewart 1932):
Formula
• 90% stock formic acid…………5-10 ml
• Formaldehyde (37-40%)………… 5 ml
• Distilled water………… to 100 ml
(A formic acid decalcifier with added formalin, claimed to fix and decalcify).13
Buffered Formic Acid (Evans & Krajian 1930):
Formula:

• 20% aqueous sodium citrate …………65 ml
• 90% stock formic acid……………. 35 ml
(This solution has a pH of approximately 2.3.An effective formic acid decalcifier
buffered with citrate).2,13
Formic acid:
Formula
• Formic acid (90%)…………..100 ml
• Distilled water…………… 900 ml
• (A simple effective decalcifier).12
Kristensen:
Formula
• Formic acid……18ml
• Sodium formate……..3.5g
• Distilled water……..82ml
(An effective formic acid decalcifier buffered with formate).12
Advantages:
• Only weak acid used extensively as primary decalcifier.
• It simultaneously fixes and decalcifies the tissue.
• It is gentler and slower and suitable for immunohistochemical staining.1,13
DISADVANTAGES:
• Delayed decalcification if the specimen size is more.

• It can still damage tissue, antigens, and enzyme histochemical staining.13
2. TRICHLOROACETIC ACID:
• Introduction:
Trichloroacetic acid (TCA; TCAA; also known as trichloroethanoic acid) is an
analogue of acetic acid in which the three hydrogen atoms of the methyl group have
all been replaced by chlorine atoms.9
• History:
The discovery of trichloroacetic acid by Jean-Baptiste Dumas in 1839 delivered a
striking example to the slowly evolving theory of organic radicals and valences. The
theory was contrary to the beliefs of JönsJakob Berzelius, starting a long dispute
between Dumas and Berzelius.14
Table 3 : Properties of Trichloroacetic acid18
Properties
Molecular formula C2HCl3O2
Appearance White solid
Density 1.63 g/cm³
Melting point 57 to 58 °C (135 to 136 °F; 330 to 331 K)
Boiling point 196 to 197 °C (385 to 387 °F; 469 to 470 K)
Solubility in water Soluble in 0.1 parts
Acidity (pKa) 0.66
Structure
Dipole moment 3.23 D

Fig 4: Structure of Trichloroacetic acid18
• Synthesis:
It is prepared by the reaction of chlorine with acetic acid in the presence of a suitable
catalyst.9
Advantages:
• Show superior results in staining and cell morphology.2
Disadvantages:
• Slower in action but lesser compared to formic acid.2
• “Acetic acid and Picric acid are weak acids which cause tissue swelling and
are not used alone as decalcifiers but are found as components in Carnoy’s
and Bouin’s fixatives”.1,2
III. CHELATING AGENTS:
These are organic compounds which have the power of binding certain metals. The
chelating agent used for decalcification is ethylene-diaminetetracetic acid (EDTA).
Its use as a decalcifying agent being first described by Hilleman and Lee (1953).
It is a slow process as calcium is removed layer by layer from the hydroxyapatite
lattice.15

Tissues decalcified by this method show a minimum of artifact and may
subsequently be stained by most techniques with first class results. These qualities
make it the decalcifying agent of choice for electron microscopy.11,15
Although EDTA is nominally acid, it does not act like inorganic or organic acids but
binds metallic ions, notably calcium and magnesium. EDTA will not bind to calcium
below pH 3 and is faster at pH 7-7.4, even though pH 8 and above gives optimal
binding; the higher pH may damage alkaline-sensitive protein linkages (Callis &
Sterchi 1998).15
EDTA binds to ionized calcium on the outside of the apatite crystal and as this layer
becomes depleted more calcium ions reform from within; the crystal becomes
progressively smaller during decalcification. This is a very slow process that does
not damage tissues or their stainability.10,15
When time permits, EDTA is an excellent bone decalcifier for immunohistochemical
or enzyme staining and electron microscopy. Enzymes require specific pH
conditions in order to maintain activity, and EDTA solutions can be adjusted to a
specific pH for enzyme staining. EDTA does inactivate alkaline phosphatase but
activity can be restored by addition of magnesium chloride.11,14
EDTA and EDTA disodium salt (10%) or EDTA tetra-sodium salt (14%) approach
saturation and can be used as simple aqueous or buffered solutions at neutral pH of
7-7.4, or added to formalin. EDTA tetrasodium solution is alkaline, and the pH
should be adjusted to 7.4 using concentrated acetic acid. The time required to totally
decalcify dense cortical bone may be 6-8 weeks or longer although small bone
spicules may be decalcified in less than a week.15

• FORMALIN/EDTA (HILLEMANN & LEE 1953)
Formula
• EDTA, disodium salt.................. 5.5 g
• Distilled water……… 90 ml
• Formaldehyde (37-40% stock)…………….10 ml.13
AQUEOUS EDTA, PH 7.0-7.4
Formula
• EDTA, disodium salt…………….. 250 g
• Distilled water……………1750 ml
If solution is cloudy, adjust to approximately 25 g sodium hydroxide. Solution will
clear.16
EDTA solution
Formula
• EDTA (disodium salt)……….. 55 g
• Formalin…………100 ml
• Distilled water…………………900 ml
EDTA is sometimes known as Sequestrene, or Versene.16
Advantages:
Causes little tissue damage.
Conventional stains are largely unaffected.2,16
Disadvantages:
Acts slowly.2

FACTORS INFLUENCING THE RATE OF DECALCIFICATION
Several factors influence the rate of decalcification, and there are ways to speed up
or slow down this process. The concentration and volume of the active reagent,
including the temperature at which the reaction takes place, are important at all
times. Other factors that contribute to how fast bone decalcifies are the age of patient,
type of bone, size of specimen, and solution agitation. Mature cortical bone
decalcifies slower than immature, developing cortical or trabeculae bone. Of all the
factors, the effectiveness of agitation is still being debated.1,2
Fig 5: Factors influencing rate of decalcification
(I) HEAT:
Increased temperature accelerates many chemical reactions including
decalcification, but it also increases the damaging effects acids have on tissue so

that, at 600
C, the bone. Soft tissues and cells may become completely macerated
almost as soon as they are decalcified. Chemical reaction is accelerated two to three
times rise of temperature, and Murayama, Suzuki, and Itoh (1937) ascertained that
with increased temperature the process of decalcification actually takes place within
an even shorter time.2,6
The optimal temperature for decalcification has not been determined. Although
Smith (1962) suggested 250
C as the standard temperature, but in practice a room
temperature (RT) range of 18-300
C is acceptable. Conversely, lower temperature
decreases reaction rates and tissues not completely decalcified at the end of working
week. A better recommendation is to interrupt decalcification but briefly rinsing
acid off bone, immersing it in neutral buffered formalin (NBF), and resuming
decalcification on the next working day.17
Microwave, sonication, and electrolytic
methods produce heat, and must be carefully monitored to prevent excessive
temperatures that damage tissue (Callis and Sterchi 1998).16
Increased temperature also accelerates EDTA decalcification without the risk of
maceration, but may not be acceptable for preservation of heat- sensitive antigens,
enzymes, or electron microscopy work. Brain (1966) saw no objection to
decalcifying with EDTA at 600
C if the bone was well fixed.17
(II) STRENGTH / CONCENTRATION OF ACID:
Generally, more concentrated acid solutions decalcify bone more rapidly but are
more harmful to the tissue. This is particularly true of aqueous acid solution, as
various additives, e.g. alcohol or buffers that protect tissues may slow down the
decalcification rate. With combination fixative-acid decalcifying solutions, the
decalcification rate cannot exceed the fixation rate or the acid will damage or
macerate the tissue before fixation is complete. Consequently, the decalcifying
mixtures should not compromise the balance of desirable effects (e.g. speed) with
the undesirable effects (e.g. maceration, impairing staining).15

In all cases, total depletion of an acid or chelator by their reaction with calcium must
be avoided. This is accomplished by using a large volume of fluid compared with
the volume of tissue (20: 1 is usually recommended), and by changing the fluid
several times during the decalcification process. Brain, however, pointed out that if
a sufficiently large volume of fluid is used (100 ml per g of tissue) it is not necessary
to renew the decalcifying agent even though depletion is less apparent in a larger
volume.17,15
Ideally, acid solutions should be endpoint tested and changed daily to ensure that
the decalcifying agent is renewed and that tissues are not left in acids too long or
over- exposed to acids, i.e. ‘over-decalcification’.18
(III)AGITATION:
The effect of agitation on decalcification is controversial even though it is generally
accepted that mechanical agitation influences fluid exchange within as well as
around tissues with other reagents. Therefore, it would be a logical assumption that
agitation speeds up decalcification and studies were done attempting to confirm this
theory.17
Russel (1963) used a tissue processor motor rotating at one revolution per minute
and reported the decalcification period was reduced from 5 days to 1 day. Others,
including Clayden (1952), Brain (1966), and Drury and Wallington (1980), repeated
or performed similar experiments and failed to find any time reduction.18
The sonication method vigorously agitates both specimen and fluid, and one study
noted cellular debris found on the floor of a container after sonication could possibly
be important tissues shaken from the specimen (Calis & Strerchi 1998).17
Gentle fluid agitation is achieved by low speed rotation, rocking, stirring, or
bubbling air into the solution. Even though findings from various studies are
unresolved, agitation is a matter of preference and not harmful as tissue components
remain intact.17, 18

(IV)SUSPENSION:
The decalcifying fluid should be able to make contact with all surfaces of a specimen
and flat bone slabs should not touch each other or the bottom of a container as this
is enough to prevent good fluid access between the flat surfaces. Bone samples can
be separated and suspended in the fluid with a thread or placed inside cloth bags tied
with thread. Some workers devise perforated plastic platforms to raise samples
above a container bottom to permit fluid access to samples.18
(V)VACUUM:
Another way to speed up the diffusion of one of the reaction products of the
decalcification process is to pump off the gaseous carbon dioxide. Waerhaug (1949)
explains the more rapid decalcification he observed by the closer contact between
decalcifying fluid and object caused by the rapid removal of the carbon dioxide gas.
Very slight differences in rates of decalcification between the usual method and that
in vacuo were found in the present series.17
When the decalcification procedure is watched in vacuo it appears that the process
is considerably accelerated, mainly due to the "turbulent" development of the carbon
dioxide bubbles. It should be borne in mind, however, that it is the low pressure that
causes the considerable expansion of these gas bubbles (at a pressure of 2 cm. Hg
the volume increases 38 times compared with that at atmospheric pressure) though
the decalcification process is not necessarily accelerated.2,17
According to Le Chatelier the reaction equilibrium is influenced by the dissolution
of calcium salts when the formed carbon dioxide is pumped off rapidly, but
could not perceive any shortening of the decalcification time.18
In opinion it is the rapidly expanding gas bubbles that prevent the decalcifying fluid
from reaching the surface of the object in an adequate amount, for the same amount
of the reaction product (Carbon dioxide) occupies a much larger part of the object
and "blocks" it from any further supply of fluid. (By "surface" not only the outer

surface is understood but also the “inner surface," namely, the surface of the spaces
containing the blood vessels as well as that of numerous osteocyte cavities) With
decrease of pressure the reaction equilibrium is shifted in favour of the rate of
decalcification, but the possibility of reaction decreases. Both these factors are
supposed to result in fairly equal times of decalcification whether in vacuo or not.1,18

METHODS OF DECALCIFICATION
• Decalcification using acids/ Manual method
• Microwave decalcification
• Ion exchange resins method of decalcification
• Electrolytic decalcification
• Ultrasonic decalcification
DECALCIFICATION METHODS USING ACID SOLUTIONS:
Acid solutions are most widely used for routine decalcification of large amounts of
bone and calcified tissue. The principle underlying the action of acid decalcifying
agents involves the solubilities of metallic salts. Calcium occurs in bones chiefly as
the carbonate and phosphate salts, and these salts are only slightly soluble in
water.2,18
An acid will act to release the calcium from its combination with the anions and
effect an ion exchange to give a soluble calcium salt. For example, when
hydrochloric acid is used as the decalcifying agent, the released calcium combines
with the chloride ion to form calcium chloride, a soluble calcium salt.16
The calcium ions released will remain in the decalcifying solution itself and are
effectively removed from the bone.17
The general technique to be followed when one
employs acid decalcifying agents is as follows:
1. Selection of tissue
2. Fixation, after fixation, the tissue is washed to remove excess fixative.
3. Decalcification

Fig 6 : Acid decalcification method3
(For best results, gauze-wrapped-bone should be suspended in center of
decalcifying fluid)
The selected tissue should be loosely wrapped in gauze and then suspended in the
center of a large jar that is filled with the decalcifying fluid of choice. About 100
times the volume of the tissue is a good approximate amount, and this large volume
is necessary since the mineral content of a good-sized piece of bone will soon
neutralize the small amount of acid present in the solution. Decalcifying fluids that
may be used include aqueous or alcoholic solutions.19
For the rapid decalcification of bone, stronger solutions of nitric or hydrochloric acid
may be used if employed in conjunction with phloroglucin.17
The phloroglucin acts,
in a way not presently understood, to prevent organic constituents of bone from
being injured by the swelling and macerating action of the strong acids. Tissue
should be removed from the decalcifying fluid as soon as the decalcification process
is complete, otherwise the histologic and cytologic detail will be harmed.18
MICROWAVE DECALCIFICATION:
Microwave decalcification is a novel technique compared to the manual method. In
this method, hard tissues are placed in the decalcifying agent in a microwave oven

for intermittent periods with regular changes of the solution till the end point is
reached. Microwave irradiation has been shown to speed up the process of
decalcification significantly–from days to hours. It has been reported that the
decalcification of bone is accelerated about 10 times compared with that at ambient
temperature.2,14
METHODOLGY: A domestic microwave oven (LG Intellowave, Model 1911HE)
with a fixed rotary plate, maximum power output of 700 W and input voltage 230
V 50 HZ used, 100 ml fresh distilled water and irradiated to maintain the
temperature at around 41-43°C. The glass beaker placed at different points in the
oven while irradiating it to determine the best position of the specimen during
microwave decalcification, since the microwave oven used had a constant timing but
not a constant temperature. All the specimens fixed in 10% neutral buffered formalin
fixative and then washed in water for about 30 min before decalcification.14
Each sample suspended in a beaker with the help of a thread in approximately 100
ml of decalcifying agent for decalcification. The exact time at the start of
decalcification should be noted. The decalcifying solutions changed and pH and
temperature of the solutions should be recorded on a daily basis.14,19
Fig 7: Microwave oven17
The idea of using microwaves to decrease the time for decalcification of temporal
bones was originally introduced by Hellstrom and Nilsson (1992) for rat cochleas.
More recently, microwaves have been demonstrated to be useful in reducing the time

needed for decalcification in EDTA of dense, primate temporal bones (Madden and
Henson, 1997).The energy produced by microwaves generated in a domestic oven
interacts with dipolar molecules by imparting kinetic energy and altering the electric
fields. This energy induces a dielectric field leading to a rapid oscillation of dipolar
molecules at about 180°C, generating heat that is rapidly distributed homogeneously
within the tissue.14,19
Pitol et al, (2007) showed there was a 30 fold increase in decalcification speed
compared to the traditional method when the material was irradiated in a microwave
oven. However, Balaton and Loget (1989) reported that the decalcification of bone
is accelerated about 10 times in the microwave oven compared with that at ambient
temperature.19
Summary:
A new method using microwave oven was seen to accelerate the decalcification. The
choice of decalcifying agent and method is largely dictated by the urgency of the
procedure. The potential application of microwave energy in histotechnology was
first recognized by Mayers (1970). This form of nonionizing radiation produces
alternating electromagnetic fields that result in the rotation of dipolar molecules such
as water and the polar side chains of proteins through 180° C at the rate of 2.45
billion cycles/second. The molecular kinetics so induced result in the generation of
energy flux which continue until radiation ceases.14,15
ION EXCHANGE RESIN METHOD OF DECALCIFICATION:
Before proceeding with this method for decalcification, one must fix and wash the
selected tissue. Following these steps, the bone is decalcified with a mixture of
formic acid and a commercially available ion-exchange resin. The calcium is rapidly
removed from the solution of formic acid into the resin, this eliminates solution
changes that must be carried out to effect proper decalcification with the acid
decalcification methods.18

Tissue is placed in a bottle in a mixture of l0% or 20% resin and formic acid.
Cancellous bone (2 to 3 mm in thickness) will decalcify in 2 to 3 hours in the solution
and thicker pieces (5 to 6 mm in thickness) will take 4 to 8 hours to decalcify.1, 2
A
40% resin and formic acid solution may be employed where speed is essential, but
for good tissue preservation, the bone should not be left in this strength solution any
longer than necessary (up to 8 days). If speed is not essential, tissue may be left in
the following solution up to 20 days without distortion of tissue.19
• WIN-3000 (ion-exchange resin)…….100gm
• 10 % formic acid (aqueous)…………..800 ml
Fig 8: Ion-exchange method (Gauze-wrapped specimen is placed on top of
resin)17
Fig 9: Ion exchange resin17

The advantages of using the ion-exchange method for bone decalcification include
well preserved cellular detail, superior to that obtained with the acid decalcification
methods; faster decalcification; and elimination of the daily solution change. In
addition, the resin, once used, may be reclaimed for further use by washing to
remove excess acid. The washing is followed by l% ammonia water wash, overnight
treatment with saturated ammonium oxalate, and final water wash the next day.2,18
ELECTROLYTIC DECALCIFICATION:
This method employs electrolysis to shorten the time required for decalcification of
bone sections. Materials used in the technique include a durable glass jar containing
the acid decalcifying solution in which is immersed the electrode assembly and bone
specimen, as shown in the figure. The bone specimen is suspended by a platinum
wire anode in the jar, and the insoluble calcium salts are changed to ionizable salts
by the action of the acid in the solution.2,20
-A recommended electrolytic decalcifying solution is as follows:
• 88% formic acid……..100 ml
• Hydrochloric acid…….80 ml
• Distilled water……..820 ml
Current, which is supplied by a power unit, causes an electric field between the
electrodes, and this enables the calcium ions to migrate rapidly from the specimen
(anode) to the carbon electrode (cathode). The acid radicals will migrate from the
cathode to the anode. The temperature of the reaction is regulated between 300
to
450
C. Temperatures exceeding the upper limit of 450
C will cause the disintegration
of the specimen.1,20
Solutions should be changed after 8 hours of use to ensure maximum speed of
decalcification. Prolonged washing of the specimen after electrolysis is unnecessary;
the tissues are rinsed well in alkaline water and the sections immersed in lithium

carbonate before staining. The lithium carbonate treatment of a cut section will
neutralize any remaining acid in the tissue so that the acid cannot interfere with any
staining procedure.19,20
The chief advantage to the electrolytic method lies in the shortened time required for
complete decalcification. This faster time will speed diagnosis and give better
preservation of soft-tissue patterns.1,19
Fig 10: Electrolytic decalcification apparatus3
Staining reactions are usually better, since the method acts fast enough and the
tissues have a relatively short time in the acid bath. Generally, cancellous bone 3 to
5 mm in thickness will decalcify in 45 minutes or less. More compact bone will take
16 hours or longer.1,2
A disadvantage to this method is that only a limited number of specimens may be
processed at any one time. Maintaining contact between tissue and electrode may
also create problems.2,19
ULTRASONIC METHOD OF DECALCIFICATION:
The unique technology of the ultrasonic decalcification offers the rapid destruction
of crystalline structures like calcium phosphate, magnesium phosphate and calcium
carbonate. In combination with adequate solutions it provides maximum cell tissue

preservation. All diffusion processes are significantly accelerated by the Ultrasonic
method.2,19
The advantage is decalcification and intermediate rising solutions work much faster,
save up to 75% decalcification time for bone marrow biopsies. The additional feature
of cooling the tissue samples at a temperature of 17 °C avoids the warming up of the
specimens resulting from the chemical reaction with the decalcifying solution. This
guarantees 100% preservation of the morphological structures and the antigenity of
the samples20
.There are no artifacts from shrinking or swelling and all consecutive
histological and immuno-histochemical methods can be applied to these samples.19
Fig 11: Ultrasonic machine17
Depending on the sample size the user can choose from inserts including 4, 9 or 49
sample containers with different volumes. In every batch up to 49 sample containers,
able to carry multiple samples, can be processed simultaneously.17,19
Decalcification of bone specimens of 2-5 mm thickness can be achieved in 5 hours
or less when the decalcifying fluids are agitated by ultrasonic energization.2,20

HISTOCHEMICAL METHODS FOR DECALCIFICATION
Standard methods of decalcification using nitric, hydrochloric, and other acids are
unsatisfactory if histochemical techniques are to be done on the tissue, since acid
treatment will destroy the enzyme activity. Analytical methods for nucleic acids and
polysaccharides should also be preceded by a histochemical, rather than routine,
method for decalcification since these substances are largely destroyed by the acids
employed in the routine technics.11,19
Histochemical decalcification methods include the use of chelating agents (EDTA)
and buffer mixtures. Calcium salts may be removed from bone when it is placed
into a buffered solution of citrate, pH 4.5. Calcium salts are soluble at this pH, and
the zinc present in many buffered solutions of citrate will produce a reversible
inactivation of the alkaline phosphatases (Subsequent reactivation will allow for
their demonstration). Daily changes of the buffer are necessary and the
decalcification progress may be checked by use of the chemical oxalate test
described in the section on acid decalcification methods.20
Tissue should first be fixed in cold 80% alcohol for 24 to 48 hours. It is then placed
in the buffer solution at refrigerator temperature (40
C) until decalcification is
complete. Tissue is then washed in tap water, followed by distilled water, and then
placed in a sodium barbital solution at 370
C for 6 hours to neutralize the tissue from
the effects of acid citrate and to reactivate the enzyme activity. After the barbital
treatment, the tissue is washed for 3 hours in running tap water, and processed for
subsequent enzyme analysis.19, 20
Solutions used are as follows:
Citric acid- ammonium citrate buffer (pH 4.5)
• 1 N citric acid (monohydrate, 7%)…..50ml
• I N ammonium citrate (anhydrous, 7.54%)……..950ml

• 1% zinc sulfate………2ml
• Chloroform……..0.1ml
Sodium barbital solution
Sodium barbital…….100ml
Glycine……….75mg
Other buffer mixtures
L Molar hydrochloric acid-citrate buffer at pH 4.5
I N hydrochloric acid…….540ml
I M sodium citrate solution………….460ml
(Use 29.4% of the dihydrate or 35.7% of the following compound)
Lorch's citrate hydrochloric acid buffer at pH 4.4
• Citric acid crystals…..14.7gm
• 0.2 N sodium hydroxide………..700ml
• 0.1 N hydrochloric acid……….300ml
• 7% zinc sulfate……….2ml
• Chloroform…………0.1ml
Acetate buffer at pH 4.5
• 1 N acetic acid……….520ml
• 1 N sodium acetate (8.2%anhydrous or 13.6% crystalline)…….480ml
• l% zinc sulfate….2ml
• Chloroform………0.1ml.18

DEMONSTRATION OF GLYCOGEN IN DECALCIFIED TISSUES
Total or partial losses of glycogen result when tissues are decalcified with acids or
EDTA. Glycogen in muscle and marrow cells will withstand decalcification best
when fixed in aqueous formalin acidified with acetic or formic acid. Thorough
fixation of protein surrounding glycogen and embedding in celloidin before paraffin
will create semi permeable membranes surrounding the glycogen and thus hinder
diffusion the following techniques for decalcification to subsequently demonstrate
glycogen.18
TECHNIQUES:
• Celloidin
a. Tissue should be fixed in acetic alcohol formalin 24 hours at room
temperature (250
C) or 3 to 4 days at 50
C. Dehydrate with alcohols and
infiltrate for 3 days with l% celloidin in equal volumes of alcohol and ether.
b. Transfer to 80% alcohol to harden celloidin.
c. Decalcify with 5% formic acid (aqueous); solution should be changed daily
until a negative chemical test is obtained.
d. Wash 6 to 8 hours in running water, dehydrate, clear, and embed in paraffin.19
• Hard protein fixative
a. Fix tissues as described above.
b. Transfer to Bouin's fluid for 3 days at 250
C.
c. Decalcify in daily changes of 10% formalin with 5% formic acid.
d. Wash 8 hours in running water; dehydrate, clear, and embed in paraffin.19,20

END POINT DETERMINATION
Because of the harmful effects of acid on tissue, they must be left in decalcifying
fluids the minimum time possible. Accurate determination of the endpoint of
decalcification is therefore necessary. Experience will enable a value judgment to be
made upon approximate time required, depending on the tissue structure and size of
the block. Small biopsies of cancellous bone should be examined after 24 hours, and
other specimens daily after two to three 24-hour changes of fluid.15
TYPES OF END POINT DETERMINATION:
• PHYSICAL METHOD
• CHEMICAL METHOD
• RADIOGRAPHIC METHOD
PHYSICAL METHOD:
• Experience will enable a value judgement to be made upon approximate time
required, depending on the tissue structure and size of the block.
• Experienced hands can tell by the 'feel' of the tissue if decalcification is
complete.
• Probing the tissue with a needle is not recommended, judicious bending or
trimming can be valuable, but this damages the tissue.
• Another method used was to test weight loss or weight gain procedure that
provides relatively good, quick results with all acids and EDTA (Mawhinney
et al. 1984, Sanderson et al. 1995).17
• Although still used, physical tests are considered inaccurate and damaging to
tissues.

• Probing, needling, slicing, bending or squeezing tissue can create artefacts,
e.g. needle tracks, disrupt soft tumour from bone, or cause false positive micro
fractures of fine trabeculae, a potential misdiagnosis.19
Bubble Test:
Acids react with calcium carbonate in bone to produce carbon dioxide, seen as a
layer of bubbles on the bone surface.
The bubbles disperse with agitation or shaking but reform, becoming smaller as less
calcium carbonate is produced.
As an endpoint test, a bubble test is subjective and unreliable but can be used as a
guide to check the progress of decalcification, i.e. tiny bubbles indicate less calcium
present.18
Fig 12: Probing needle17
Fig 13: Weighing machine17
CHEMICAL METHOD:
• This method depends upon the identification of calcium in the decalcifying
solution.
• It therefore follows that the endpoint can only be detected by sampling the
fluid change following completion.
• This method cannot be used following EDTA decalcification

Calcium oxalate test (Clayden 1952):
This method involves the detection of calcium in acid solutions by precipitation of
insoluble calcium hydroxide or calcium oxalate, but is unsuitable for solutions
containing over 10% acid even though these could be diluted and result in a less
sensitive test.20
Solutions:
1. Ammonia hydroxide, concentrated.
2. Saturated aqueous ammonium oxalate.

Method:
Fig 14: Methodology of calcium oxalate test17
Result:
• If a white precipitate (calcium hydroxide) forms immediately after adding the
ammonium hydroxide a large quantity of calcium is present, making it
unnecessary to proceed further to step 3 which would also be positive.

• Testing can be stopped and a change to fresh decalcifying solution made at
this point.
• If step 2 is negative or clear after adding ammonia hydroxide, then proceed to
step 3 to add ammonium oxalate.
• If precipitation occurs after adding the ammonium oxalate, less calcium is
present.
• When a smaller amount of calcium is present, it takes longer to form a
precipitate in the fluid, so if the fluid remains clear after 30 minutes, it is safe
to assume that decalcification is complete.19,20
RADIOGRAPHIC METHOD:
This is the most sensitive test for detecting calcium in bone or tissue calcification.
The method is the same as specimen radiography, using a FAXITRON with a
manual exposure setting of approximately 1 minute, 30 kV, and Kodak X-OMAT
X-ray film on bottom shelf. It is possible to expose several specimens at the same
time.19
The method is: Rinse acid from sample, place carefully identified bones on
water-proof polyethylene sheet on top of the X-ray film, expose according to
directions, and leave bones to place until film is developed and examined for
calcifications.1,2
Bones with irregular shapes and variable thickness can occasionally mislead on
interpretation of results. This problem is resolved by comparing the test radiograph
to the pre-decalcification specimen radiograph, and correlating suspected calcified
areas with specimen variations.2
Areas of mineralization are easily identified, with tiny calcifications best viewed
using a hand-held magnifier.1

Radiography only indicates the presence of deeper foreign objects and care must be
taken during microtomy not to damage the knife.18
Fig 15: Stages of end point determination by radiographs2
SURFACE DECALCIFICATION:
• Once decalcification is complete, surface acid should be washed from the
tissue with water. If there is any delay before processing the tissue should be
returned to formal-saline. Although there are advocates of neutralizing the
acid before processing it is probable that it will all be washed out by the
processing fluids.14
• Occasionally, an unexpected area of calcification may only become apparent
whilst a paraffin-wax block is being trimmed. If only a small area is involved
it is possible to decalcify the surface layer by inverting the block in 5%
hydrochloric acid for 1 hour or so.2
• This is less drastic than returning the tissue to aqueous solutions. As only the
top 30 m or so is likely to be decalcified, care should be exercised to collect
the first sections cut. Before cutting, the block should be rinsed in water to
avoid contaminating knife or microtome with acid.1,2

ARTIFACTS IN DECALCIFICATION
Impacted bone fragments:
When samples of bone are taken for histological examination, either by needle or
trephine, or when using a saw to remove a small sample from a large specimen, the
bone is subjected to significant trauma which often leads to displacement of bony
fragments and disruption to the adjacent soft tissues.
Remedy: Trim deeply into the specimen to avoid the most traumatized areas close
to the surface.8
Bone dust artifact:
Undecalcified sections may contain a fine grit- like, bone dust or powder seen
within or in close proximity to the bony trabeculae. This material is more apparent
in H & E stained sections where it stains strongly with hematoxylin but is largely
obscured by von Kossa staining. When deposited in the bone marrow it stains black
with von Kossa, suggesting it originates from the calcified trabecular matrix. The
artifact is probably produced during sectioning and becomes more prominent with
increasing section thickness
The bone dust occurs in sections prepared with both glass and diamond knives, and
there appears to be no reliable way of preventing it.8

Fig 17: Bone dust artifact8
Effects of over-decalcification on tissues:
Most artifacts in decalcified tissues relate to poor initial fixation, over fixation, or
incomplete decalcification. Artifacts arising from the first two situations are
irreversible whereas incomplete decalcification can be rectified by surface
decalcification of the paraffin block.
Specimens subjected to decalcification by mineral or organic acids must be
protected from the hydrochloric action of these agents by proper fixation. Digestion
of cellular and other tissue components occurs more rapidly when the tissue is
unfixed or only partially fixed. Prolonged exposure to acid decalcifying agents will
eventually damage even well fixed tissues, so determining the end point of
decalcification accurately is essential in all situations. Over decalcified sections
typically stain strongly with eosin and show a marked loss of nuclear
hematoxyphilia. Nuclear and cytoplasmic detail is poorly preserved.8

Fig 18: Over decalcification in a section of bone8
Effects of incomplete decalcification:
Incomplete decalcification of specimen is clearly apparent when trimming a paraffin
block. As discussed previously, this problem is readily corrected, but there are
associated problems such as damage to the microtome knife and to the soft tissue
surrounding the calcified areas. If sections can be obtained, the bony trabeculae stain
strongly with hematoxylin (indicating residual calcium) and the soft tissue is
severely disrupted.8
Fig 19: Incomplete decalcification in a section of bone8

Limitation of paraffin wax embedding for dense bone:
Paraffin wax may not adequately support dense bone and resultant sections may
show holes, folds and coarse chatter. Soaking the trimmed face of the paraffin block
in a softening agent for 5 to 15 minutes prior to chilling may improve the special
quality.8
Fig 20: Paraffin section of decalcified dense bone showing holes, folds and
coarse chatter8
PRECAUTIONS TAKEN DURING STAINING:
• Use freshly prepared hematoxylin
• Routine hematoxylin stain time should be doubled
• Acid differentiation step should be shortened
• Bluing solutions should be mild bases to avoid loss of bone structure caused
by ammonia after bluing.
• Collagen stains to demonstrate mature and fine immature fibres in certain
tumours and fracture callus.18,20

CONCLUSION
Decalcification is a straightforward process but to be successful requires:
• A careful preliminary assessment of the specimen
• Thorough fixation
• Preparation of slices of reasonable thickness for fixation and processing
• The choice of a suitable decalcifier with adequate volume, changed regularly.
• A careful determination of the endpoint
• Thorough processing using a suitable schedule.

REFERENCES
1. Callis GM, Bancroft JD. Theory and Practice of Histological Techniques 6th
ed. Edinburgh: Churchill Livingstone. 2008;338-360.
2. Culling CF, Allison RT, Barr WT. Cellular Pathology Technique 4th ed.
London: Butterworths.1984;408-30.
3. Callis G, Sterchi D. Decalcification of Bone: Literature Review and Practical
Study of Various Decalcifying Agents, Methods, and Their Effects on Bone
Histology. The Journal of Histotechnology 1998;21:49-58.
4. Prasad P and Donoghue M. A comparative study of various decalcification
techniques. Indian journal of dental research 2013;24:302-8.
5. Sanjai K, Kumarswamy J, Patil A, Papaiah L, Jayaram S and Krishnan L.
Evaluation and comparison of decalcification agents on the human teeth.
Journal of Oral and Maxillofacial Pathology 2012;16:222-7.
6. Afreen Nadaf MS, Radhika MB, Lalita J, Thambiah, Paremala K, Sudhakara
M. Decalcification: a simpler and better alternative. Journal of Dentistry and
Oral Bioscience 2011;2:10-3.
7. Mathews B J, Mason G I. Influence of decalcifying agents on
immunoreactivity of formalin-fixed, paraffin embedded tissue.
Histochemical journal 1984;16:771-787.
8. Rolls GO. Difficult Blocks and Reprocessing. Leica Microsystems 2011.
9. Joshi H, Parikh N, Soni N, Jain S. Comparison of Efficacy of Various
Decalcifying Agents on Human Teeth. Journal of research and advancement
in Dentistry 2014;3:47-52.
10.Lillie, R. D. Histopathologic technique and practical histochemistry, ed. 3,
New York, McGraw- Hill Book Co,1965.

11.Zappa J, Cieslik-Bielecka A, Adwent M, Cieslik T, Sabat D. Comparison of
different decalcification methods to hard teeth tissues morphological analysis.
Dent Med Probl 2005;42:21-26.
12.Verdenius H W and Alma L. A quantitative study of decalcification methods
in histology. Journal of Clinical Pathology 1958;11:229-236.
13.Athanasou N A, Quinn J, Heryet A. Effect of decalcification agents on
immunoreactivity of cellular antigens. Journal of Clinical Pathology
1987;40:874-878.
14.Sangeetha R, Uma K, Chandavarkar V. Comparison of routine decalcification
methods with microwave decalcification of bone and teeth. Journal of
Maxillo-Facial Pathology 2013;17:386-399.
15.Mattella Grando L, Westphalen L, Bento Pelisser V, Vieira F et al.
Comparative analysis of two fixating and two decalcifying solutions for
processing of human primary teeth with inactive dentin carious lesion. Revista
Odonto Ciencia 2007;22(56):99-105.
16.Mawhinney WHB, Richardson E, Malcolm AJ. Technical methods control of
rapid nitric acid decalcification. J Clin Pathol 1984;37:1409-1415.
17.Morse A: Formic acid – Sodium Citrate Decalcification and Butyl Alcohol
dehydration of teeth and bones for sectioning in teeth and bones for sectioning
in paraffin. J Dent Res 1945;24:143-153.
18.Myers, R.L. The 100 most important chemical compounds: a reference guide.
Greenwood Publishing Group. 2007;141-147.
19.Warshawsky H, Moore G. A technique for the fixation and decalcification of
rat incisors for electron microscopy. J Histochem Cytochem 1967;15:542-9.
20.Waerhaug J. Decalcification of bone and teeth under vacuum- a rapid method
for producing hard tissue preparations. J Dent Res 1949;28:525.

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