3 Decalcification and

,'

other treatments for

hard tissues

11. Decalcificatien by acids

"

45

II Decalcification by cheloting agent$

33.0eCIIlciticlltioD in praclicll
311 AciII dllC8lcifien

46
41
.

~B

3.3.2 Ole\alio, Mm EDrA

3.3.3. Erld'poIm 01 de!:alc,fcation

49
49

3.4. Softenilg of nl)ll-<:IIIClIreGU mlllarials .. "

3.5. Softening specimens already embedded in WIX

50
51

When the hard mineral eomponents are the sale objects of interest, a thin seaion
of a dry piece of bone O'" tooth can be prcpared by grinding (see Culling. 1974;
Dickson, 1984). This is a skilled and time-consuming procedure that wastes most
of the piece being grOOM. but il provides preparations in which the mineral crystals are clearly VIsible (see sanderson, 1997). Hard specimens canflot be sectioned

with an ordinary microtome, but they can be softened after flXation, usualty by
rernoving the substances responsible for lhe hardness of the lissue. Decaloficalion
is the chemical dissolution of insoluble caldum S<!lts with a suilable aad 01 chelating agent. Other approaches are available far softening h~d materials lhat contain
silica, o( which awe their hardness lo campad organic materials.
Tlssues such as bone, dentine, horn and wocd can be embedded in plastic
(Chapter 4) and cut with a particularly rugged mi::rotome, equipped with a chiselshaped knile of hardened steel. This is me lechoaogy of choice lO laboratories that
specialize in such fields as bone maturation and wood anatomy. Techniques lar
bone and teeth are thoroughly discussed by Callis (2002) and AA and Martin
(2004), and methods loc hard p1ant tissues are descnbed in Berlyn and Miksche
(1976) and Ruzin (1999).

l.1. Decalcification by acid.

lhe prindpal mineral component al the calcified tissues of verlebr~te anlmals is a
hydroxyapatite formally designated as Ca lO(P0 4);(OHh uke omer 'insoILble' salts
Ihis exists, when wet, in equilibrium wilh its salurated soIution, whlch contains very
low concentrations of calcium, phosphale, and hydroxide lons:

Continuous removal of cakium, phosphate, (l( hydroxide ioos from the so'ution will
preverlt the system from reaching equi~brium. TIle readion will therefore proceed
from leh to right until all the h~roxyapatite has dissolved. If the liquid surrounding
the spedmen has a high concentration of hydrogen ions, \he reaction:

WlII be dnven from left to right, removing the hydroxide ions liberated as a result of
dissolution of the hydroxyapatite. Any strong acid could serve as a source of hydrogen ions, but Ihose that form sparingly soluble calcium salts (e.g. sulphuric acid)
are, far obvious reasons, unsuitable. Hydrochloric, nitric and formic acids are Ihe
ooes most often used. The overall readions of lhese adds with hydroxyapatile are
respectively:
Ga

(P0 4 )6(OH)2 + 20H+ + 20cr

1O

10Ca2+ + 20Cr + 6H P0 + 2H 0
3 4
2

The calcium from the tissue ends up as calcium ions dissolved In the decalcifying
fluid. If the latter is changed frequently the completion of decalcification can be recognized when extraded calcium ions are no longer delectable by a simple cnemi
calles!. .
The aystallanice of the hydroxyapatile of bones and teeth inrorporates small numbers of carbonate ions in addition to the more abundant phosphates and hydroxides. The mineralized tissue contains, in effect. a small percentage af calcium
carbonate. This is dissolved by acids:

Minute bubbles 01 carbon dioxide are fQ(lTled within and on Ihe surfaces of specimens being decalcified in adds, but they do not usually produce signs of damage
visible under the microscope.
Mast enzymes are put out of aaion by acid decalcifying agents, but the strudure
of the tissue is only slightly disrupted provided that fixation has been adequate.
Immunohistochemical stilining is often poor after decalcification ilt km pH, and
weilker acids such as acetic (Henzen-Longmans el al., 1965) or ascorbic, pH aboul
2.5, (Merchan-Perez el al., 1999) are preferred for most antigens.
Decalcification by ilcids can result in hydro/ysis of nudeic adds. RNA is broken into
soluble fragments, V\lth resulting redudion or 1055 of cytoplasmic staining by cationic dyes. The purine and pyrimidine bases are removed from !he sugar-phosphilte
backbone of ONA; this occurs less completely than in a deliberille Feulgen hydro/ysis (Chilpter 9), bul nevertheless inlerferes with the interpretation of densitometric
measurements of histochemically stained sections Of with in situ hybridiziltion (A1es
el al., 1999). In a comparison of decalcifying solutions Shibata el al. (2000) found
that hybridization of labelled probes with mRNA in dental tissues was severely
impaired by mineral acids but was satisfac:l.ory alter soIutions containing formic aeid.

3.2. Decalcificalion by chelating agenls

A chelating agent is an organic compound or ion that is able to combine with a
metal ion to fQ(lTl a compound known as a metal chelate. In the chelate, the metal
atom is covalently boulld as part of a 5- or 6-membered ringo Chelates are stable
compounds and do nol readily decompose to liberate metal ions. Consequently,
the reaaion of a metal ion with a chelilting agent, although reversible, proceeds
almost to completion if the chelating agent is present in excess.

3.3 Decalcification in pradice l.!!J

Ethylenediamine tetraacetic acid (EDTA) forms ordinary salts (four are possible)
with sodium and other alkali metals, but the ethylenediamine tetraacetate ions
combine with most other metal ions to form stable, soluble chelates. If a piece of
calcified tissue is immersed in liquid containing EDTA anions, free calcium ions will
be removed from the solution by chelation. Hydroxyapatite will therefore dissolve
because it will be unable to attain equilibrium with a saturated solution.

[EDTA]2 As in a solulion of lhe

disodium sall of EDTA

The bonds to lhe Ca alom are of

equal lenglh and mutually at
right angles, directed as if to the
vertices of a regular octahedron.

For a full account of the chemistry of chelation, see Chaberek and Martell (1959).
lhe chelation of metal ions by dye molecules is described in Chapter 5. of this
book. Decalcification by EDTA differs from decalciflcation by acids in that hydrogen
ions play no part in the chemical reaction involved. lhe chelating agent is used on
the alkaline side of neutrality, so the deleterious effects of acids on labile substances such as nucleic acids and enzymes are avoided. Strongly alkaline solutions
are not used, however, because they extract proteoglycans from the extracellular
matrix of the tissue (lppolito et al., 1981). The main disadvantage of EDTA is that
it acts more slowly than the acids.
Note that EDTA has many names, including versene, sequestrene, edetic acid, ethylene-bis(iminodiacetic acid) and (ethyjenedinitrilo)tetraacetic acid, with corresponding names for the socIium salts. In a solution of any of the salts the number of
ionized carboxyl groups available for chelation increases with the pH. A 1% solution of Na2EDTA has a pH of 5.3 whereas with Na3EDTA the pH is 9.3 and with
Na 4 EDTA it is 11.3.

, H. Decalcification in practice
Specimens \hat are to be decalcified must be properly fixed and no longer responsive to changes in osmotic pressure. Usually the flXative must be thoroughly
washed out of the tissue with water prior to decalcification in order to avoid undesirable chemical reactions. For example, residual sodium phosphates from a
buffered formaldehyde flXative would oppose the action of an acid decalcifier.
Dichromate ions would be reduced by formic acid, and mercury or zinc would form
a chelate with EDTA. lhe volume of decalcifying fluid should be at least 20 times
that oi the specimen. An acid mixture is changed every 24-48 h; an EDTA solution

l 48 I Chapter 3 IDecalcficaton and other treabnents for hard tssues

every 3-5 days. If the ammonlum oxalate test (Section 3.3.3) is to be used, the
anticipated last change of decalcfer should have only about five times the volume
of the specirnen, so that any calcum in the liquid will be present at higher concentration than in a larger volume.
Teeth present various difficulties not encountered with bone. The enamel consists
almost entirely of calcium salts, with a tenuous framework of protein that usually
collapses after decalcification. The fixative should be one that strongjy cross-links
protein molecules; glutaraldehyde is suitable. lhe pulp can shrnk, tearing the odontablast processes, which pass from the pulp into the dentine. lhis artifact is also
reduced by glutaraldehyde fixation (van Wyk, 1993).

3.3.1.
Add decaldfiers

Formic acid, at pH 1.5 to 3.5 is the decalcifier of choice for most purposes. Several
mixtures similar to the one below have been described. Sorne of them also contain formaldehyde. in an attempt to offset the consequences of inadequate primary
fixation. ther organic acids used for decalcifcation indude acetic and ctric, which
are slow, and lactic, which is nearly as fast as formic (Eggert and Germain, 1979).
Adequately fixed specimens (severa I days in formaldehyde; shorter times in more
rapidly acting fixatives) can be decalcified in 1.0 M hydrochloric or nitric acid
(Chap:~r 20). lhese mineral acids (pH = O) remove calcified deposits more quickIy than formc acd (see Sanderson, 1994). De Castro's fluid is one of many older
decalcifying fluids that contain a mineral acid. Ascorbic acid is suitable only for delicate objects containing only thin layers of bone.
An acid decalcifier typically takes 4-5 days to soften a 5 mm cube of cancellous
bone (Culling, 974). Nilsson et al. (1991) perfused mts with a flxative, followed
by an acid decalcifier for 6 h. This was as effective as immerslng fixed tissues for
n h in the same liquid. A more generally applicable way to reduce the time consists of heating to 55C in a microwave oven. lhis is said to produce a lO-fald
acceleration of the reaction (Kok and Boon, 1992). lhus, a pece of bone requiring 30 h to decalcify at room temperature can be ready to dehydrate and embed
after as little as 3 h in at 55C.
When decalcification is complete (Section 3.3.3) the speClmen should be washed
in severa! changes of water or 70% alcohol.

Buffered formc acd (C1ark, 1954)

Formic acid (90%):
Water:
Sodum formate (HCOONa):

250 mi
750 mi
34 g

Alternatively, a solution with the same compositlon can be made by dissolving

19.8g of sodium hydroxide in 729 mi water and adding 271 mi of 90% formic acid.
This solution keeps ndeflnrtely. Its pH is 2.0, and it produces only minimal suppression of the stainability of nucleic acids. Ippolito et 01. (1981) found that formic acid
at pH 2.0 extracted less proteoglycan from cartilage than 5 other decalcifying
agents tested.

De Castro's fluid
Absolute ethanol:
Chloral hydrate:
Water:
Concentrated nitric acid (70% HN0 3):

300 mi
50 g
670 mi
30m!

Mix in arder stated. Keeps Indefinitely.

Cauton. The nitric acid must be added last. Concentrated HN03 reacts explosively
with concentrated ethanol.

3.3 Decalcification in praetice

49

De Castro's flUid is traditionally used in conjunction with neurological staining methods. It is strongly acidic and decalcifies rapidly. The alcohol and chloral hydrate in
the mixture are supposed to prevent swelling of the tissue, presumably by ncreasing the osmotic pressure of the fluid. This may be unimportant when adequately
flxed specimens are being decalcified. However, de Castro's mixture was formerly
used as a simultaneous fixing and decalcifying agent In this circumstance the
osmotic effects, as well as the fixative properties of al! three ngredents, assumed
greater slgnificance.

Ascorbc ocid n so/ne (Merchan-Perez et a/" 1999)

Ascorbic acid:
Sodium chloride:
Water:

1.0 g
0.85 g
100ml

This solution is prepared immediately before use and is used only once. It is suitable for small specimens, notably the inner ears of small animals, with which
Mercha n-Perez et a/. (1 999) found im munohlstochemical staini ng for various antlgens to be superior to that seen after decalcfication in EDTA.

3.3.2
CheJation with
EDTA

Decalcification wlth EDTA proceeds much more slowly than wth acids, several
weeks occasiona!1y being required, but this is not Injurious to the t;ssues. EDTA s
frequently used when the sectlons are to be stained with immunohistochemical
methods, and s able to unmask some antgens (Hosoya et a/., 2005; see also
Chapter 19). After decalciflcation with EDTA some histochemical methods for
enzymes can subsequently be performed upon frozen sedions. An EDTA solution
is made up immediately before using and is used only once.

Dsodum EDrA solution

Thls solution is recommended for routine decalciflcation.
Either 5 or 10 g of disodlum ethylenediamine tetraacetate, [CH 2 N(CH 2 COOH)
CH 2COONa h.2H 20, is dissolved in 100 mi of water, and 4% NaOH is added until
the pH is between 7 and 8.

Ammonum EDrA soluton

This solution is recommended for specimens containing dense bone. Sanderson et
al. (1995) found that t took 6 days to decalcify material that required 18 days in
other EDTA mxtures.
Ethylenediamine tetraacetc acid [CH 2 N(CH 2COOH)2b:
(Note that this is EDTA acid, not one of its salts.)
Ammonium hydroxlde (ammonla solution, 28-30%, SG 0.9):
Water:

14 g

9 mi
76 mi

Stir until dissolved, then add more ammonium hydroxide until the pH is 7. 1.
The specimen is put in a perforated plasbc casette in the jar contaning the EDTA
solution, and left on a magnetic stirrer to keep the liquid In gentle motion while
decalcificatlon proceeds. An EDTA soluton should be changed every third or fourth
day. when deca!cified, the specimens should be washed in water befare dehydration, because the EDTA salts are insoluble In alcohol. (Alcohol-soluble salts of EDTA
are avallable, but accordi ng to Eggert et o" (1981) they have no special adva ntages
as histological decalcifying agents.)

3.3.3.
End-point of
decalcificaton

If the specimen is large enough, extends beyand the reglan of interesl and contains calcified tissue throughout, it may be trimmed with a razor blade or scalpel at
intervals during decalciflcatian. When it can be cut easlly, it will also be 50ft enough
to be sectioned on a microtome. Sometimes the specimen is too small to be
trimmed, ar the calcifled material is isolated In the middle of the block. An

l 50

Chapter 3

IDecaicification and other treahnents for hard tissues

othelWise unwanted piece of bone of approximately the same size can then be
processed alongside the specimen. When this piece of bone is fully softened the
specimen for hlstological study should also be decalcified. Specimeos should never
be tested by poking needles into them; the holes will persist in the sectons.
A more exaet test for completeness of decalciflcation makes use of the facl that calcium oxalate, though soluble in mineral acids, is insoluble in water aod io aqueous
solutions of alkahs.
Ca Z+ +

eZo42-

HZO

--+-

The lest is conducted as follows:

(1)

(2)

Add drops of strong ammonia solulion (ammonium hydroxlde, se 0.9) to

about 5 mi of used decalcifyi ng fluid until the mixtu re becomes alkall neto litmus paper (pH > 7).
Add 5 mi of a saturated aqueous solution of ammonium oxalate (approximately 3% (NH4)2C204.H20; can be kept 00 the shelf for years) and leave
to stand for 30 min.

l no white precipitate has formed after this time, the fluid contains no calcium ions.
Thls test can also be used to determine the end-point of decalcification by EDTA,
even though solutions of the latter do not contain free calCium ions (Eggert and
Germain, 1979). lhe [caEDTAF- anion presumably dissociates as the highly insoluble oxalate is formed. Rosen (1 981) recommends adjusting EDTA solutlons to pH
3.2-3.6 for maximum sensitivity of the oxalate test.
If an X-ray machne is available, me presence or absence of calcified deposits in a
speClmen can be demonstrated by radiography. Control specimens known to contalO and not to contain calcified materia I should be X-.rayed alongside the speClmen
being testE;d. in order to assess the amount of radio-opacity due to soft tissues.
It.is p~~larIY importa~t t? determine the end-poi~t of decalcificaton when 1..0 M
n1tnc 'or hydrochlonc aCld IS used, because excesslve exposure to these solullOns
will cause hydrolysis of proteins and macromolecular carbohydrates as well as
nudeic acids, with consequent struetural damage.

3.4. Softening af non-calcareaus materials

Cartlage in vertebrates and chtin in arthropods are composed largely of macromolecular carbohydrates, but they often also contain insoluble calcium salts. lhese
materials can be softened to some extent by demneralizaton in acids or chelating
agents. The hard external layer of insed cuticle can be softened with an alkahne
hypochlorite solution and removed by careful dissecton (Haas, 1992).
Wood is cellulose, reinforced by lignin (Chapter 10), and aften contains deposits
of silica (Si0 2). Crystals of silica can cause sectioning difficultes with other tissues,
including leaves of some grasses. Hydrofluoric acid (HF), whlch dissolves silica, is
sometimes used as a softeoing agent for hard plant tissues. The concentration may
range from 15% to 60% depending on the hardness, applied for 12-36 h (see
Ruzin, 1999). If calcium silicate is also present, the solution should also include a
little sulphuric aCld (Tomasi and Rovasio, 1997). Extreme cauton is needed when
using concentrated hydrofluoric acid: protectlon of the eyes, hands (nitrile or natural rubber gloves) and body (natural rubber or neoprene apron) and an adequate

3.5 Softening specimens already embedded in wax

I 51

fume hood. Calcium carbonate or hydroxide powder should be to hand for neutralizing spills. Calcium fluoride is insoluble and not hazardous. Many institutions
require specific training of personnel who are to use HE
Carlqust (1982) preferred a solution containing ethylenediamine. Its mechanism
of adion is uncertain. The original author used 4% ethylenediamine, prior to desilicifying very hard woods with 24% HF (Kul<achka, 1977; this publication gives
detailed instructions and is freely dvailable on the internet).

Ethylenediamine solution lor softening wood (Corlquist, 1982)

Ethylenediamine [H2N(CH2)2NH2l:
Water:

10 mi
to make 100 mi

Caution. Elhylenediamine is a caustic alkali. Keep stoppered to reduce absorption

of atmospheric CO 2 .
Put boiled or alcohol-nxed wood in the ethylenediamine solution far 3 days.
Dehydrate and clear in t-butano! (Chapter 4) and embed In paraffin wax. Berlyn
and Miksche (1976), Carlquist (1982) and Ruzin (1999) provide many other usefui pradical instrudions for processlng hard plant tissues.

3.5. Softening specimens already embedded in wax

Incomplete dehydration or clearing is one cause of excessive hardening of normalIy soft tissues embedded in paraffin, but specimens are sometimes unexpectedly
difficult lo cut for no discernible reason. Hard material encountered at the time of
sectionng can be softened by cutting sedions of the trimmed wax block unt"tl the
tissue is entered, and then Immersing the face of the block in water for 15 to 30
mino Water enters the exposed tissue, and aften softens it for a depth of up to
500 IJm into the block.
Softenlng agents containing ethanol, glyeerol and phenol are preferred to water by
many workers, and were found to be supenor to plain water In a comparatlve study
by Wynnchuk (1992). A suitable mixture was Horne's soften~g agent, with the
following composition:
~
95% Ethanol:
Glycerol:
Acetone:
Lquid phenol (this is an 80% aqueous soluDon):

476 mi

160 mi
40 mi
40 mi

For sorne other softening agents that are easily made in the laboratory, see
Sanderson (1994). If the water or softening mixture is cold (G-4"C) it will harden
the wax, which also facilitates sectioning.