THE JOURNAL OF HI8TOCHEMISTRY AND CYTOCHEMI8TRY Vol. 17, No.

2
Copyright © 1969 by The Histochemical Society, Inc. Printed in U.S.A.

ON THE HISTORY AND MECHANISM OF ALIZARIN AND ALIZARIN

RED S STAINS FOR CALCIUM”2

HOLDE PUCHTLER, SUSAN N. MELOAN AND MARY S. TERRY

Department of Pathology, Medical College of Georgia, Eugene Talmadge Memorial Hospital,

Augusta, Georgia
Received for publication October 18, 1968

Alizarin (madder) has been used in textile dyeing since early antiquityc In histology cal-
cium-alizarin or calcium-alizarin red S compounds are often referred to as “lake” or “com-
plex.” Chemical and infrared spectroscopic data showed that these compounds are salts, not
chelates. In dye chemistry the term lake denotes a poorly soluble or insoluble salt of a
water-soluble dye. Salt formation between calcium deposits in tissues and alizarin or aliza-
rn red S is indicated by the sensitivity of these compounds toward dilute acetic acid. Acid
dyes for lakes, which do not contain chelating groups, also stained calcium deposits selec-
tively. Alizarin stained calcium deposits intensely only around pH 12. Alizarin red S colored
calcium deposits selectively around pH 9; neutral and acid dye solutions produced severe
diffusion artifacts. Chemical data indicate that alizarin red S can react with calcium via its
sulfonic acid and/or its OH groups.

In histology alizarin red S and von Kossa’s been referred to as a lake or complex. Further-
technique are commonly used for demonstration more, since the publication of the reviews by
of calcium salts. Von Kossa’s procedure is Harms (22) and McGee-Russell (32), new
believed to visualize phosphate and carbonate chemical and infrared spectroscopic data on
anions, whereas alizarin red S reacts with calcium calcium-alizarin-alizarin red S and related com-
and other cations. Both methods have been used pounds have become available. It was therefore
for diagnosis of calcium deposits. However, deemed of interest to reinvestigate the mechanism
during investigations of early arteriosclerotic of alizarin and alizarin red S stains for calcium
lesions we observed striking discrepancies be- and to search for a method which would avoid
tween the amounts of “calcium” demonstrated diffusion artifacts. Since this project was part of
by alizarin red S and by von Kossa’s technique. a study of early arteriosclerotic lesions, investiga-
For example, in some arteries von Kossa’s tions were limited to calcifications in soft tissues;
procedure colored segments of the internal bone and teeth were excluded. These studies and
elastic membrane black, yet adjacent sections a review of pertinent literature are presented in
utterly refused to bind alizarin red 5, and vice this report. Observations on the mechanism of
versa. Obviously, for an understanding of the von Kossa’s procedure will be published sepa-
processes occurring in these arteriosclerotic rately.
lesions, it was essential to obtain information
MATERIALS AND METHODS
concerning the histochemical significance of these
Experiments were carried out on human autopsy
procedures. A preliminary perusal of the literature
material. Blocks of tissues were fixed in absolute
showed differences between chemical and histo-
alcohol, Carnoy’s fluid (absolute alcohol-chloro-
logic concepts; e.g. chemists classify the calcium-
form-glacial acetic acid, 6:3:1), 10% unbuffered
alizarin or alizarin red S compounds as a salt, formalin or Zenker-formol (Spuler, Maximow)
whereas in histology and histoehemistry it has and embedded in Paraplast by Autotechnicon.
Sections were cut at 5 p.
‘Dedicated with admiration to Dr. R. D. Lillie,
One batch of alizarin siccum (Chroma, C.I.
who by his far ranging knowledge of the history
and chemistry of dyes, elevated staining from a 58000), four batches of the biologic stain alizarin
craft to a branch of histochemistry. red S (Chroma, C.I. 58005), one sample of the
2 This investigation was supported by a grant- corresponding textile dye Diamond red W (Verona
in-aid from the Georgia Heart Association and Dyestuffs) and one sample of alizarin blue S
United States Public Health Service Research
Grant HE 12147 from the National Heart Insti- (Chroma, C.I. 67415) were used. Solutions, 0.1
tute. and 0.5%, of the sulfonated dyes in distilled water

110
 ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM 111

and in 0.5% aqueous solutions of Brook’s buffers, fast scarlet FGN (0.1. 18020), levanol yellow 6G
pH 4.8, 7.2 and 9.0 were prepared. To determine (C.I. 23900), Niagara sky blue 6B (0.1. 24410),
possible effects of the buffer salts on the staining orange G and wool orange 2G (0.1. 16230), Sirius
reaction, Sorensen’s phosphate buffer and barbital supra red 4BLA (0.1. 29065), Sirius supra tur-
buffer, pH 7.2 and 9, were substituted for Brook’s quoise LG (0.1. 74180), Solophenyl orange TGL
buffer. Owing to the poor solubility of alizarin, (0.1. 40215 and 40220) and tartrazine 0 (0.1.
only saturated solutions of this dye in pH 7.2 and 19140). Sections were stained in 0.5% solutions
9 buffers and in 0.3 and 1% aqueous NaOH were of these dyes for 5 mm or 1 hr.
tested. Sections were stained for 5 mm or 1 hr, The dye names given above are the trade names
rinsed in buffer solution for about 5 sec, dehy- used by the manufacturers. Previous paper chro-
drated in three changes of absolute alcohol, matographic studies showed significant discrep-
cleared in xylene and mounted in Permount. Other ancies between the composition of some dye
series were stained with alizarin red S as recom- samples carrying the same Colour Index number
mended by McGee-Russell (32). obtained from different manufacturers (44). It
To obtain information concerning the selec- was therefore deemed expedient to use the trade
tivity of alizarin and alizarin red S for calcium, names of the dyes employed rather than syno-
the reactivity of various salts was studied in nyms. Other brands of these dyes may or may not
model experiments. To avoid lengthy repetition, yield similar staining patterns.
the compounds used are itemized only in Table I. Calcium and related salts were removed from
Approximately 500 mg of the substance (A.C.S. sections by treatment with 5% aqueous acetic
reagent grade) to be studied were placed near acid for 5 or 10 mm. Sections were then washed
one end of a slide. Two to 3 drops of the dye solu- in distilled water and stained as described above.
tion were added; another drop was placed at the Since alizarin-metal chelates are insoluble in
other end of the slide to serve as a color standard. acetic acid, whereas salts of alizarin are readily
The interaction between test substance and dye soluble (22), stained sections were treated with
solution was observed between parallel and 5% aqueous acetic acid for 5 mm to obtain in-
crossed polaroids for 2-5 mm or until no further formation concerning the nature of the bond
changes occurred. This procedure is a modification between dyes and calcium deposits in tissues.
of the technic recommended by McGee-Russell Paper chromatograms of the four batches of
(32) for staining of calcium in tissue sections. alizarin red S and of Diamond red W were pre-
In another series the salts were placed on slides, pared according to the method described by
mixed with gelatin, air-dried, fixed in formalde- Rosenthal, Puchtler and Sweat (44).
hyde vapor or Carnoy’s solution and stained as A Reichert Zetopan microscope equipped with
described above for tissue sections. a twin lamp unit (tungsten and mercury vapor
To investigate the role of the sulfonic acid lamp), bright field and dark field condensers, was
group of alizarin red S in the staining of calcium used for comparison of staining and fluorescence
deposits in tissues, the staining patterns of sal- microscopic patterns. The combination of ultra-
fonated dyes without chelating groups recom- violet blue pass filter BG 12/3 mm and ultraviolet
mended by Pratt (38) or the Colour Index (10) blue barrier filter Sp. 3 (GG 9/1 mm + OG 1/1.5
for preparation of barium or calcium lakes were mm) was employed. Dyes which were not fluores-
studied. The following dyes were employed: cent under these conditions were studied also
Bordeaux red B (C.I. 16180), brilliant black (C.I. with ultraviolet pass filter UG 1/1.5 mm and
27260), croceine orange Y (C.I. 15970), fast light barrier filter Sp. 2 (GG 13/1 + 3 mm + Wratten
rubine BL (C.I. 17065), fast red S (C.I. 15620) and foil 2B).
Ponceau 2R (C.I. 16150). To determine whether The term “calcium deposits” is used in a de-
or not high affinity for earth alkalis is a peculiarity scriptive sense without strict chemical denota-
of certain acid dyes, a series of sulfonated dyes tions; these areas may or may not contain other
(without chelating groups) was selected for com- cations in addition to calcium.
parison, namely Acilan cosine E (C.I. 14710),
Acilan red S2B (C.I. 23910), amaranth (C.I. RESULTS
16185), azophloxin GA and fast crimson GR (C.I.
18050), azorubin S (0.1. 14720), Benzo fast scarlet Effect of fixatives: Comparison of sections
4BS (C.I. 29160), Benzo fast scarlet 4GS (C.I. from the same organ fixed in the solutions listed
29185), Biebrich scarlet WS (0.1. 26905), brilliant above and stained under identical conditions
scarlet 6R (C.I. 16255), croceine scarlet 6R (C.I. showed striking differences. Coloration of calcium
16255), croceine scarlet MOOP (0.1. 27290), di- deposits was most intense in alcohol- and Carnoy-
phenyl green GPD (C.I. 30295), durol black 2B fixed sections; no difference was observed between
(0.1. 26370), fast wool red GL (C.I. 17045), levanol these two fixatives. In formalin- and Zenker-
112 PUCHTLER, MELOAN AND TERRY

fixed material calcium deposits were weakly to around calcium deposits were already severe in
moderately colored. Staining of calcium deposits sections stained for 5 mm. Individual granules,
decreased with the duration of storage in formalin e.g., in muscle fibers or renal epithelial cells, were
and was abolished after 2-3 weeks. The staining not recognizable; the cells were colored uniformly
properties of material fixed in Zenker-formol orange-red.
varied significantly and seemed to depend on the Alizarin red S in buffer solutions, pH 4.8:
duration of fixation and subsequent washing. The staining patterns obtained with these solu-
Therefore, unless otherwise stated, all descriptions tions were very similar to those produced by
and comments hereafter refer only to alcohol- and unbuffered aqueous solutions.
Carnoy-fixed tissues. Paper chromatograms of the old batch of alizarin
Binding of alizarin: Alizarin was practically red S and of Diamond red W were very similar.
insoluble in buffers of pH 7.2 and did not stain The dye moved in a well defined band which was
calcium deposits. Saturated solutions of alizarin framed by traces of bluish pink material. The
in buffers of pH 9 or in 0.3% aqueous NaOH other three dye samples contained less alizarin
colored most calcium deposits moderately; very red S and showed significant amounts of yellow
small deposits were only faintly stained. Very impurities which moved more slowly than the
intense bluish red to purple coloration of all dye; the dye band was not homogeneous but
calcium deposits within 5 mm was obtained with included a brownish substance. The pH values of
saturated solutions of alizarin in 1% aqueous 0.1% aqueous solutions of these dyes were: old
NaOH; other tissue structures remained un- batch of alizarin red S, 3.5; Diamond red W, 3.7;
stained. Small intracellular deposits were clearly other batches of alizarin red 5, 2.7, 2.9 and 2.9;
delimited and there were no diffusion artifacts. thus the impurities were apparently more acid
Sections stained with this procedure were used as than alizarin red S.
standards in assessing the specificity and intensity McGee-Russell’s procedure: Under the condi-
of coloration obtained with other staining tech- tions of McGee-Russell’s procedure (32) all dye
niques described below. Unfortunately, in this samples stained calcium deposits deep orange.
strongly alkaline solution sections tend to become Intensity and hue of background coloration varied
detached from slides and the method is therefore with the amount of impurities in the dye samples,
inconvenient for general use in hospital pathology. as described above for 0.1% aqueous solutions of
Attempts to substitute 1% NaOH in 70% ethanol these dyes. When staining time was limited to 1
as a dye solvent were unsuccessful; under these mm, diffusion of the orange substance was moder-
conditions calcium deposits were only weakly to ate. However, small intra- or extracellular gran-
moderately colored, even when staining was ules, which stood out clearly in adjacent sections
prolonged up to 1 hr. stained with alkaline solutions of alizarin, alizarin
Aqueous unbuffered solutions of alizarin red S or alizarin blue S (see below), could not be
red S: The staining properties of 0.1% solutions of identified; such areas were colored uniformly
alizarin red S varied widely from batch to batch. orange.
One batch, which had been purchased several Alizarin red S in buffer solutions, pH 7.2:
years before, yielded deep orange-red coloration of Calcium deposits were colored orange-red. Sec-
calcium deposits within 5 mm; other tissue tions stained for 5 mm showed moderate diffusion;
structures were stained light pink. When staining granules of calcified material in cells and muscle
time was extended to 1 hr, the intensity of colora- fibers were not distinguishable. All other tissue
tion of calcium deposits was strikingly decreased structures were stained faintly pink; binding of
and other tissue structures were moderately the yellow impurities in three dye batches was
stained. This loss of contrast made it difficult to abolished. However, some yellow coloration
identify small foci of calcification. Practically persisted in and around calcium deposits, particu-
identical staining patterns were obtained with the larly between closely spaced lesions. In sections
corresponding textile dye Diamond red W. A stained for 1 hr the intensity of coloration of
sample of alizarin red S bought in the course of calcium deposits and background staining were
this study stained calcium deposits yellowish moderately increased, and diffusion around
orange; all other tissue structures were colored calcified areas was more marked.
bright yellow (staining time 5 mm). Two further Alizarin red S in buffer solutions, pH 9: In
samples from different batches of alizarin red S sections stained for 5 mm calcium deposits were
yielded similar staining patterns. No trace of the stained red without a yellow tinge; all other
red dye could be found in sections stained for 1 structures were unstained or were colored faintly
hr in the three dye samples containing yellow pink. Even small granules of calcified material
impurities; tissues were colored uniformly yellow. stood out clearly against the practically colorless
With all dye samples tested, diffusion artifacts background. No trace of yellow could be found in
 ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM 113

these sections; the staining patterns obtained selective dyes varied from 6.2 to 9.5 and were
with the different dye batches were identical. comparable to those of acid dyes for lakes.
Intensity of coloration of calcium deposits in- In sections stained with Ponceau 2R the strong
creased with time of staining up to 1 hr. No halos red fluorescence of calcium deposits contrasted
or other signs of diffusion were found in this well with the faint greenish gray primary fluores-
series; even in sections stained for 1 hr calcium cence of other tissue structures. Calcium deposits
deposits were sharply delineated, e.g., in renal stained with Bordeaux red B were weakly to
epithelial cells or cardiac muscle fibers containing moderately fluorescent. Sections treated with
numerous granules. brilliant black, croceine orange Y or fast light
Alizarin blue S: Solutions of this dye in rubine BL did not show secondary fluorescence.
distilled water or in pH 7.2 buffers stained fairly Dyes which produced background staining were
large calcium deposits weakly; small deposits were unsuitable for fluorescence microscopy. For
barely recognizable. Calcium deposits were example, azorubine 5, which produced slight to
moderately colored after staining for 5 mm in a moderate background staining, rendered calcium
0.5% solution of alizarin blue S in pH 9 buffers; deposits intensely fluorescent, but other tissue
optimal coloration of calcium deposits were structures showed strong secondary fluorescence.
obtained by staining for 1 hr. Under this condition Treatment of sections with acetic acid: No
calcium deposits were colored deep blue to reddish calcium deposits could be found in sections pre-
blue and were sharply delimited. All other tissue treated with 5% aqueous acetic acid for 5 or 10
structures remained unstained. mm and stained with alizarin, alizarin red S or
Acid dyes for lakes: In sections stained for 5 acid dyes for lakes; other tissue structures were
mm, the sulfonated azo dyes Bordeaux red B, faintly colored or remained unstained. When
brilliant black, croceine orange Y, fast light stained sections, which showed intensely colored
rubine BL and Ponceau 2R stained calcium de- calcium deposits, were treated with 5% aqueous
posits selectively. Intensity of coloration was acetic acid for 5 mm, this coloration was removed.
increased when staining time was prolonged to 1 Occasionally a few large calcified areas remained
hr. Diffusion did not occur; calcium deposits were faintly stained.
sharply delineated. Excellent contrast between Model experiments: Dispersions of various
intensely colored calcium deposits and practically calcium and other salts in gelatin were found
unstained tissues was obtained with Bordeaux unsuitable. When such model slides were stained
red B, brilliant black and fast light rubine BL. in acid dye solutions, the fairly acidophilic gelatin
The deep yellow coloration imparted by croceine also bound the dye and evaluation of the reactions
orange Y and the yellowish red color of Ponceau of embedded salts became difficult, if not im-
2R were found less convenient for studies of possible. Alkaline dye solutions (pH 9-12) caused
fine granules in early stages of calcification, e.g., swelling of the gelatin, and membranes of gelatin
in arteriosclerosis and in renal tubular epithelial tended to become detached for the slides. These
tells. The pH value of the 0.5% aqueous solutions series were therefore regarded as unsuitable for
of these dyes ranged from 7.0 (brilliant black) to critical evaluation.
9.7 (Bordeaux red B). Solutions of these dyes In contrast, when the dye solutions were added
buffered to pH 4.8-5 stained all tissues strongly to salts on a glass slide under microscopic control,
and identification of calcium deposits became changes of color were easily observable. The
difficult or impossible. results of this series are summarized in Table I.
Aqueous 0.5% solutions of azorubin S (pH 6.5), Obviously, alizarin and alizarin red S were not
Biebrich scarlet WS (pH 6.2), fast red S (pH 6.2) specific for calcium, nor for earth alkalis, but
and croceine scarlet MOOP (pH 9) stained calcium reacted with a wide variety of cations. Further-
deposits intensely; other tissue structures were more, the colors of the dye salts were also not
weakly or moderately colored. The staining specific for certain cations. The formation of
patterns produced by these dyes closely resembled colored dye salts depended, at least in part, on
those obtained with aqueous solutions of alizarin the solubility of the compounds tested. For
red S at pH 5 or below. Contrast between calcium example, the practically insoluble BaSO4 did not
deposits and tissues decreased with prolongation react with the dye solutions; in contrast, the
of the staining time beyond 5 mm, owing to soluble BaCh produced significant color changes.
increasing background staining. The other sal- The color changes of the dyes commenced at the
fonated dyes tested colored tissues and calcium surface of the crystals. As the crystals dissolved,
deposits more or less uniformly within 5 mm. the metal-dye compounds diffused into the sur-
Selectivity of sulfonated dyes for calcium deposits rounding solution. A strongly colored layer was
was independent of the pH of the dye solution; formed on the surface of poorly soluble crystals,
the pH values of 0.5% aqueous solutions of non- but the interior of the crystals remained unstained
 TABLE I
Staining of Various Salts by Alizarin Red S and Alizarina

Salt
Alizarin Red Alizarin Red Alizarin 1% NaOH,
S,pH4.8 S,pH9 pHll.8

CuCl dR dR h R h, dOll-BR dP-RV

CuCl22H2O P h BP YR-BR decol
CuSO45H2O dRV dR RV V
CuSO4(NH4)zSO46H:O PV, B PV, B PV, B sl PV, B
CuCl22NH4Cl RV RV RV R
MgSO47H2O - dR dR dBR
MgCO3.Mg(OH),.nH,O dRsR dIlsR dR dRsR
NH4ClMgCl2 - dRaR dRsR dR.sR
MgCl26H2O - dRsR dRsR dRaR, dBR
CaCl,2H2O P dBR BrR R
CaCO, YR BR sl R dVR
CaC2O4H2O YR, R p dR BrR dBRV
CaSO42H2O YR p, h dV BR V
Ca(H,P04)2.H,O Y h R h Y Y
CaHPO4 Rh dBR BrR V
Caio(OH)2(P04)6 YR dR BR dV, Br
SrCO3 Ph - - -

SrCl26H,O OR dR p BrY RB p
BaCl22H,O YP dRsR YO dV p
BaSO4 - - - -

ZnCO, R-P si 0 - PBr

ZnC1, P p dR dR YR
CdC1, - dR dR RV p, 0-BR
3CdSO48H2O - dR dR V h
HgCl: - BrY - dBr
HgSO4 - Lighter 1Y Y
Al,(SO4),.18H20 dY OR OR p OR
Al2(S04),.18H,O OR V-OR Y, 0 BR p, 0
CaSO42H20
SnCI,#{149}2H,0 R p R p R p B-OR
SnCl45H20 Y Y Y Y
PbCO1 YRh - - BRp
PbC12 OR h dR BrV Br p
BiCl, Br Br Y Y
MnCl24H2O dY dR dBR BrR
MnSO4.H20 - P, BR p 0-BR BR p
FeCl24H20 B p P-Y B, R, Y Y
FeCl, - slBR,Y - Y
FeNH4(SO4)2 12H2O BrV p si R RV h R
Fe,(S04), (about 6H20) - BrV BrV h R h
FeCl,2NH4ClH,O - - BrV R-Br
FeSO4.7H20 RV RV V V p
Fe(NH4)2(S04)26HzO BrV dR RV p V p
CoOl, 0 dBR d.BR R, YEt, V, G
CoSOg7H,0 - dBR dBR-V R h
NiCl,6H,O P R, B R, B BR p
Ni(C,H,O,),.4H,O dBR dBR BR BP
Cr(NH4)(S04)2.12H,O RO-dR BrR BrR h Gy p
CrCls6H,O[Cr(H,O)6JCl, Br p dBr p Br p Br p
Cr(C,H,0,),.H,0 dR dR h Br h V p
K,Cr,O7 R, Br p - RBr p Y-R-Br, RBr p

#{149}
The abbreviations used are: B, blue; Br, brown; G, green; Gy, gray; 0, orange; P, pink; R, red;
Ra, rose; V, violet; Y, yellow; d, dark; h, halo; 1, light; p, precipitate; sl, slight; decol, decolorized;
-, no reaction.
114
 ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM 115

or retained the original color. Thus the dye solu- dyeing are the Leyden Papyrus X and the
tions apparently did not penetrate the crystals, Stockholm Papyrus, which are believed to have
but reacted only with the superficial layers. been written about the end of the 3rd century
Frequently, such salts resembled some calcium
A.D. (6). However, some of these recipes refer
deposits in tissues which also showed an intensely
to originals of 300 B.C. at the latest and probably
colored periphery and weakly colored or unstained
much earlier (14). The Stockholm Papyrus (7)
center. The progressive breakdown of crystal
structure and simultaneous formation of colored contains numerous mordanting procedures and
compounds could easily be followed by alternate recipes for dyeing with madder and other natural
observation between crossed and parallel polar- dyes. Lime water and alum dissolved in vinegar
oids. A few compounds did not dissolve during (aluminum acetate?) are frequently recom-
the period of observation; only a thin colored mended as mordants. For dyeing a rose color,
layer was formed on their surface. This group wool was smeared with ashes, washed in liquid
included CaC,04, SrCO, and PbCO,. from potter’s clay and mordanted; after being
A few compounds, e.g., MgCl,, did not react
rinsed in salt water, the wool was dyed in a
with alizarmn red S at pH 4.8, but produced color
solution of madder, bean meal and white oil in
changes with the other dye solutions tested.
rain water, and aftertreated with alum. To
Ca(H,P04), reacted slightly with alizarin red S
in pH 9 buffer solution but not with alizarin red obtain a purple coloration, textiles were treated
S in pH 4.8 buffer solution or with alizarin. Since first with a blue dye and then mordanted and
it appeared possible that this phenomenon might dyed with madder as described above, except
be due to a lowering of the pH of the dye solution that the concentration of madder was doubled.
by the dissolving salt (see “Discussion” below), However, it was already known that the shade
2-3 drops of the dye solvents were added to crys- of madder dyeings could be varied by the use of
tals of Ca(H,,P04), and the approximate pH of different mordants (14). Postmordanting with
the resulting saturated solution was determined alum was recommended to prevent fading; this
by indicator papers. The pH values were 2-2.5.
procedure seems to be a precursor of the after-
chroming or coppering procedures of modern
DISCUSSION
textile industry to improve fastness properties.
The literature on alizarin and alizarin red S According to Caley (7), the methods described
stains for calcium deposits in tissues has been in these papyri were essentially the ones used
reviewed comprehensively by Harms (22) and during the next 1500 years until the advent of
McGee-Russell (32). These histologic procedures synthetic dyes.
were originally based on ancient traditions in Parenthetically, the dyeing of wool on live
textile dyeing with madder (18). The term “lake” animals, commented upon so disapprovingly by
for the alizarin-calcium compound was ap- Pliny, is mentioned in the Stockholm Papyrus,
parently derived from half-forgotten early chemi- which gives “Book 3 of Africanus” as reference.
cal concepts. It therefore seems appropriate to The animals were washed and areas to be dyed
review briefly the history of dyeing with madder were mordanted with a solution of alum in
and alizarin. Explanations of the chemical vinegar. According to Pliny (23), the dyes were
mechanism of ali.zarin and alizarin red S stains, applied “by the means of certain barks of a foot
whether the term lake implies complex or salt and a half long dipped in these colours, and so
formation, are scanty. This discussion therefore imprinted and set upon their fleeces as if riotous
is limited mainly to chemical aspects of the wantonness and superfluitie should force Nature’s
interaction of these dyes with calcium and other worke, and make wool grow of the colour.”
cations. However, it seems much more probable that
History of madder dyeing: Madder was these decorations were simply proprietary im-
used for dyeing of textiles in early antiquity. prints; dyes are still used for this purpose in the
According to Forbes (14), the oldest samples are Basque region when the sheep are taken to the
pieces of cotton from Mohenjo-Daro dating mountains for summer grazing. Since alizarin on
back to the third mifiennium B.C.; in Egypt, an aluminum mordant has light fastness 7-8 (9),
textiles dyed with madder were found in tombs it seems well suited to withstand fading on
of the period of the XVIII-XXth Dynasties. animals exposed to the strong sunlight of Egypt.
The oldest extant samples of recipe books for In contrast to branding, this ancient procedure
116 PUCHTLER, MELOAN AND TERRY

did not interfere with later conversion of the TABLE II

skins to leather. Dissociation Constants of 1- and i-OH
Madder was widely distributed in the Old Groups of Anthraquinones (p4)
World and cultivated near Rome in classical
antiquity (46). The history of madder dyeing in 2-Hydroxyanthraquinone 24 X 10-’
Alizarin, first constant 6.6 X 10’
Europe has been reviewed by H#{252}bner (23) and
1-Hydroxyanthraquinone 3.2 X 10’
Hailer (21). According to H#{252}bner (23), the role
Alizarin, second constant 1.1 X 10-12
of aluminum in madder dyeing was recognized by
Petty in 1667, who concluded “that the use of
allum is to be a Vinculum between the Cloth and salts only with the 2-OH group; alkali and earth
the Colour.” During the following centuries alkali hydroxides reacted first with the 2-OH and
madder dyeing apparently came under the then with the 1-OH group of ali.zarin. Only the
influence of alchemy. To “animalize” textiles, 1-OH group participated in complex (chelate)
repeated treatments with cow, sheep or goat formation; the 2-OH group remained free and
dung prior to mordanting and dyeing and an could form salts. On the basis of these observa-
aftertreatment with dung were considered es- tions Pfeiffer (37) suggested that in dyeing with
sential by Bancroft in 1813 (23). These alchemical madder or alizarin aluminum, iron, chromium
practices were challenged less than a century ago and similar metals form a complex with the
by Schlieper and Baum, who recommended 1-OH and 9-CO group; calcium and other earth
mordanting wite aluminum and calcium salts and alkalis form a salt with the 2-OH group. The
dyeing in a solution of alizarin and calcium salts physical chemistry of alizarin and other anthra-
(21), a procedure similar to the methods de- quinone derivatives was studied by Huttig (24).
scribed in the Stockholm papyrus. The im- Comparison of the dissociation constants of
portance of calcium salts in dyeing with madder alizarin with those of 1- and 2-hydroxyanthra-
was already recognized by Hausmann in 1791 quinone indicated that the 2-OH group is more
(21), but the chemical mechanism of this calcium strongly acid than the 1-OH group (Table II), as
effect on the dyeing of alum-mordanted material already suggested by Pfeiffer (37). Correlating the
with madder or alizarin was finally clarified only physical-chemical data with the experiences of
in the 20th century. textile dyers, Huttig (24) suggested that salt
Early chemical studies of alizarin-calcium formation of a free hydroxy group with calcium
compounds: The history of alizarin was re- plays a major role in the formation of the
viewed by Graebe and Liebermann (20). Alizarin calcium-aluminum-alizarin compound. This con-
and purpurin were first isolated from madder clusion was confirmed by Attree and Perkin (2),
roots by Colin and Robiquet in 1826. Incidentally, Venkataraman (48), Harms (22), Gerstner (16)
the name alizarin was derived from alizari, the and Kiel and Heertjes (25, 26).
term for roots of Rubia tinctorum imported from Early histologic uses of madder and
the Orient. The composition of alizarin was alizarin: The literature from 1567 to the early
determined independently by Strecker and by 20th century on coloration of bones and/or
Graebe and Liebermann in 1868 (20). The teeth of animals fed madder has been reviewed
calcium, barium and lead salts of alizarin were by Cameron (8). The recognition of the relation
clearly described by Graebe and Liebermann (20). between coloration of bone by madder and
Perkin (35) published a detailed study of the Na, staining of calcium has been ascribed to Gottlieb
Na,, K, K, and Ca salts of alizarin and of the in 1914 (8). However, according to Haller (21),
role of the 1- and 2-OH groups in salt formation; Bancroft noticed this relation in 1818 and tried
he was apparently the first to suggest that in to use calcium salts-without alum-as a
alizarin-aluminum-calcium compounds calcium mordant in madder dyeing. The affinity of
“neutralized” the 2-OH group. These early madder for calcium salts of bones was known also
authors distinguished clearly between calcium to 19th century histologists. In a discussion of
salts of alizarin and the lakes (chelates) of this Lieberkuhn’s paper (1874, original not available),
dye with aluminum. Gierke (17) stated explicitly that after pigeons
The interactions of alizarin and other anthra- had been fed madder the dye combined with the
quinones with metals and bases were studied in calcium salts of bones, but not with the organic
detail by Pfeiffer (36, 37). Weak bases formed matrix; the latter could be removed by boiling
 ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM 117

in NaOH without loss of coloration (“Nach seems to have been derived from Pfeiffer’s (37)
Futterung von Tauben mit Krapp verband sich term “mixed lakes” (gemischte Lacke) for calcium-
der Farbstoff mit den Kalksalzen der Knochen aluminum-alizarin and similar compounds (5).
und nicht mit der organischen Grundsubstanz However, Pfeiffer’s (37) sharp distinction be-
derselben. Man kann diese daher durch Kochen tween calcium salt and aluminum, iron or
in Natronlosung entfernen, ohne dass die Farbung chromium chelate in these compounds became
leidet.”). blurred in the histologic literature, as shown by
Alizarin was apparently first used as a histo- Becher’s (5) complaints that in histology the
chemical reagent by LieberkUhn in 1874; he terms mordant and lake were not based on a
injected a 5% neutral solution of alizarin and rational definition and were used much more
reported reaction of the dye with calcium phos- loosely and vaguely than in the textile industry.
phate but not with calcium carbonate (17). Becher (5) tried to introduce the distinction
Despite these observations, Gierke (17) recom- between alizarin-calcium salts and chelates with
mended alizarin only for staining of spinal cord. aluminum and other metals into histology, but
In the chapter on calcium salts in the Enzyklopa- apparently with little success. In the more
die der mikroskopischen Technik, Magnus (30) recent histologic literature available to us, this
discussed the role of calcium salts in textile difference is stressed only by Harms (22).
dyeing with alizarin and stated explicity that Recent chemical and infrared spectro-
calcium forms a salt with alizarin, yet, in the scopic studies of alizarin: A rather complicated
chapter on alizarin, Magnus (30) remarked that structural formula of the calcium-aluminum-
this dye was of little use in histologic technique; alizarin compound (Fig. 1) was suggested
alizarin red S was mentioned as a stain for by Rutishauser in 1940 (22, 45). Parenthet-
neuroglia, mitochondria and spinal cord. How- ically, a similar structure has been suggested
ever, Roehl (43) obtained intense violet coloration for carmin, a calcium-aluminum compound of
of calcium deposits in kidneys with alizarin in a carminic acid (22). However, in this structure
solution of NaOH; for staining with alizarin red S (Fig. 1) there would be a large strain in the
he recommended addition of NaOH, ammonia or central bridge between the two aluminum atoms
lithium carbonate to the dye solution. These (25). Kiel and Heertjes (25-28) therefore re-
early authors apparently regarded the calcium- investigated the composition and structure of
alizarin compound as a salt. The term lake for compounds formed by alizarin and its 3-deriva-
the alizarin-calcium compound in tissue sections tives with calcium, aluminum and various other

iLo-c0-o
o. ,o ___ 0

H2OmAI 0-Ca---’O A 1”H20

/\
00 H20 00

Ca-0’

0
Fzo. 1. Formula of the calcium-aluminurn-alizarin compound suggested by Rutishauser (22,45).
118 PUCHTLER, MELOAN AND TERRY

Ca++
H0-AI#{149}” H20 ‘‘H20
,,% H20

IIIIIIX:r
Fia. 2. Formula of the calcium-aluminum-alizarin compound suggested by Kiel and Heertjes (25
on the basis of chemical and infrared spectroscopic studies.

TABLE III
infrared Absorption of Alizarin Salts and Complexes (p7, 8)

Absorption Frequencies

Compound
2-OH 1-OH 10-CO 9-CO Complex Motic

cm’

Alizarin 3400 3100-2800 1659 1630 1583, 1456

K-alizarin 3450 1623 1581, 1462
Ca-alizarin 1612 1575, 1470
Ca-Al-alizarin 1640 1525 1588, 1460
3-NO,-alizarin 3440 3050 1678 1647 1590, 1452
Ca-3-NO,-alizarin 1649 1590
Ca-Al-3-NO,-alizarin 1652 1505 1578, 1472
Alizarin red S - -#{176} 1700 1670 1618, 1462
Ca-Al-alizarin red S 1650 1534 1618, 1470

a Not given. Unfortunately, data for Ca-alizarin red S were not included by Kiel and Heertjes (27,28).

metals. Analytical data showed that the calcium- ring. These conclusions were supported by
aluminum alizarin compound contained one atom infrared spectroscopic studies (Table Ill) (27,
of calcium, one atom of aluminum and two 28). Furthermore, in the NaCl region the spectra
alizarin residues (Fig. 2) (25). In other series of potassium-aluminum-alizarin, barium-alumi-
aluminum was replaced by iron or chromium; num-alizarin, Sn(IV) -aluminum-ali.zarin, calcium-
sodium, potassium, barium or tin was substituted chromium-alizarin and calcium-iron-alizarin were
for calcium. These compounds showed the same identical with that of calcium-alurninum-alizarin,
proportions as calcium-aluminum-alizarin (26). indicating that these compounds all have es-
Electrolytic experiments demonstrated that sentially the same structure (Fig. 2); i.e., they
calcium isbound in the molecule by an ionic bond, are ionized salts of aluminum-, iron- or chromium-
whereas aluminum, at least partly, is bound by a alizarate (26).
covalent bond; i.e., it forms part of a chelate The reaction of alizarin with the chelate-form-
 ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM 119

TABLE IV 3450 cm’, an 0-H stretching frequency. Since

Maxima of Absorption Bands in Ultraviolet this band could not be due to water, it had to be
and Visible Spectrum (p8) ascribed to the 1-OH group. Kiel and Heertjes
(28) therefore concluded that in potassium 2-
Compound Absorption bands
alizarate the 1-OH group no longer forms a
‘nih hydrogen bridge with the 9-CO group, or the
Alizarin 332 (she) 434 bridge has been considerably weakened. This must
K-alizarin 350 570 be caused by salt formation at the 2-OH group.
K,-alizarin 332 (sh) 593 637 Since the 2-OK group is in conjugation with
Ca-alizarin 365 580 625 (sh) with the 10-CO group, the frequency of the
Ca-Al-alizarin 329 512 latter group must be lowered, and the band at
3-N02-alizarin 328 425
1551 cm’ has therefore been ascribed to stretch-
K-3-NO2-alizarin 351 (sh) 458
ing of the 10-CO group. The bathochromic shift
K,-3-NO2-alizarin 361 (sh) 529 568 (sh)
of the absorption bands of alizarin in the visible
Ca-3-NO2-alizarin 364 520 562 (sh)
spectrum after salt formation is also ascribed to
a Shoulder. this resonance (Table IV) (28).
The formation of the calcium salt of alizarin
ing metal aluminum alone was exceedingly slow. is shown in figure 3 (28). The proposed structure
Furthermore, the intensity of the 0-Al absorption of calcium-alizarate is supported by the following
band was much lower than that of the 0-Al band spectroscopic data. In the visible region the
in the other compounds studied. The extremely spectra of calcium-alizarate and K2-alizarate
slow reaction of alizarin with aluminum was are similar (Table IV); since the two K ions
ascribed to the strong hydrogen bond between can only be bound to the 1- and 2-0- groups,
the 1-OH group and the 9-CO group; for a the bivalent Ca should be similarly bound.
complex to be formed, this hydrogen bond has to Because calcium-alizarate shows only one
be broken or at least weakened. This occurs when stretching frequency at 1612 cm’, both -0-
salt formation at the 2-OH group takes place Ca bonds must be equivalent. Because no
before chelate formation (26, 28). These chemical bands could be found at about 1525 cnc’ (com-
and spectroscopic data also explain the ex- plex ring vibration) and at about 3500 cm (OH
periences of textile dyers that hard water or stretching frequency), formation of a calcium-
addition of chalk is essential in alizarin (Turkey alizarin complex (chelate) could be ruled out (28).
Red) dyeing. According to Becher (5) and Harms These spectroscopic observations confirm earlier
(22), in the absence of calcium alizarin stains chemical findings discussed above, which indi-
tissues mordanted with aluminum yellowish, and cated that the alizarin-calcium compound is a
the stain is not fast (ganz unecht); obviously, salt (2, 19, 35-37).
chelate formation does not take place under this It may be objected that the marked color
condition. change of alizarin upon interaction with calcium
Kid and Heertjes (28) also studied the or various other cations indicates complex forma-
structure of potassium and calcium salts of tion rather than salt type linkages. Valko (47)
alizarin in the absence of chelate-forming metals. ascribed the color change upon salt formation to
The infrared spectrum of the monobasic salt the polarizing effect of the calcium ion. As
potassium 2-alizarate had an absorption band at already mentioned, Kiel and Heertjes (27)

0 OH

Ca + +H20

Fio. 3. Formation of the calcium salt of alizarin suggested by Kiel and Heertjes (28) on the
basis of spectroscopic data.
120 PUCHTLER, MELOAN AND TERRY

demonstrated that such bathochronic shifts are example, Ca(H2PO4)2 did not form colored
due to alterations of the resonance system of compounds with alizarin in pH 9 buffer solutions
alizarin during salt formation (28) . Moreover, or in 1 % NaOH (Table I). As already mentioned,
alizarin and alizarin red S are polygenetic dyes; saturated solutions of Ca(H2PO4)2 in these
i.e., the color of the dye salt varies with the solvents had pH values of 2-2.5. Obviously, the
cation (31, 38, 40, 48). Color differences among hydroxyl groups of alizarin are not dissociated
various metal compounds of alizarin and alizarin under these conditions and salt formation cannot
red S were observed also in the model experi- take place. If a calcium deposit should consist
ments described above (Table I), but these color entirely of Ca(H2PO4),, it may remain unstained.
changes do not permit conclusions whether Hence, alizarin cannot be depended on to visualize
certain metals are bound by salt type linkage or primary calcium phosphate.
by chelate formation. Madder versus alizarin: Differences between
Correlation of chemical data and staining madder and synthetic alizarin in vital staining
properties of alizarin: As shown in Table II, of bone have been stressed by Richter (42).
the dissociation constant of the 2-OH group of According to the Colour Index (9), madder
alizarin is 6.6 x 10-’ and that of the 1-OH group contains six dyes in the form of their glycosides,
is 1.1 x 10” (24); the corresponding pK values namely alizarin (rubierythric acid, CI. 75330),
are 8.18 and 11.96 respectively. Therefore, xanthopurpurin (1 ,3-dthydroxyanthraquinone,
alizarin in aqueous solutions of pH 7 or below is C.I. 75340), xanthopurpurin-3-carboxylic acid
only slightly dissociated and shows little tend- (C.I. 75370), rubiadin (1 ,3-hydroxy-3-methyl-
ency to form calcium salts. This also explains anthraquinone, C.I. 75350), purpurin (1,2,4-
the faint coloration of bones in in vivo experiments hydroxyanthraquinone, C.I. 75410) and pseudo-
observed by Richter (42). Differences between in purpurin (purpurin-3-carboxylic acid, C.I. 75420).
vivo staining with alizarin and madder are Comparative in vivo studies with alizarin and
discussed below. In solutions of alizarin adjusted purpurin-3-carboxylic acid showed that only the
to pH 9 about 50% of the 2-OH groups should be latter gave the bright carmine red color typical
dissociated; the red coloration of calcium de- of madder-stained bones; alizarin tinted bones
posits by such solutions indicates formation of the slightly pale bluish pink; extraction of madder-
monoalizarate. Around pH 12 nearly all 2-OH stained bones confirmed that coloring of bone
and about half of the 1-OH groups are dissociated was mainly due to purpurin-3-carboxylic acid
and the blue dializarate is formed. The reddish (42). In in vitro experiments, purpurin-3-carbox-
blue coloration of calcium deposits suggests ylic acid formed colored calcium salts in the acid,
that the red monoalizarate and the blue di- neutral and alkaline range (42). Unfortunately,
alizarate may occur side by side. That the xanthopurpurmn-3-carboxylic acid and purpurmn-3..
calcium-alizarin compound in tissue sections is a carboxylic acid were not available for this study.
salt, and not a chelate, is indicated by its sensi- However, the data provided by Richter (42)
tivity toward dilute acetic acid. As pointed out indicate that the staining properties of purpurin-
by Becher (5), Cameron (8) and Harms (22), 3-carboxylic acid are similar to those of alizarin-3-
calcium salts of alizarin dissociate readily in cold sulfonic acid. Thus, staining patterns obtained
acetic acid, whereas alizarin-metal chelates with madder should be compared with those
disintegrate only after prolonged boiling in produced by alizarin red S rather than by
H2SO4 or other mineral acids. alizarin.
Unfortunately, alizarin is not specific for Interaction of alizarin red S and other
calcium but can react also with other metals sulfonated dyes with calcium: The 1- and
(Table I). In “blind” tests the color differences 2-OH groups of alizarin red S show the same
among various alizarin-metal compounds were behavior toward calcium and various other
found insufficient for identification of the cations. metals as those of the parent dye alizarin (27, 28).
Identification is complicated also by the fact Differences between frequencies of certain bands
that the anion of the salt can affect the color of in the visible and infrared spectrum of salts of
the alizarin-metal compound as shown by the alizarin and its 3-substituted derivative have
calcium and iron salts in Table I. This effect been ascribed to effects of the electronegative
seems to be due, at least in part, to alterations of nitro or sulfonic acid group (27). However, the
the pH of the dye solution by the anion. For interaction of alizarin red S with calcium is not
 ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM 121

limited to the 1- and 2-OH groups; the sulfonic acid and neutral media only the sulfonic or
acid group also forms a salt with calcium. One carboxylic groups take part in salt formation
calcium atom can react with two sulfonic acid (42). Acid and neutral solutions of alizarin red
groups; i.e., it can bind two dye molecules of S are ochre yellow; the salts (lakes) formed with
alizarin red S according to the general formula: calcium in model experiments and in calcified
2 dye-SO3Na + CaC12 - (dye-SO,)2Ca + 2 tissues are colored orange or yellowish red.
NaCl (4, 40). Salt formation between alizarin In alkaline solutions the hydroxyl groups partici-
red S and calcium, strantium, barium, zinc, pate in salt formation (42); the calcium salts
magnesium, aluminum and other metals later formed under these conditions are deep red to
than group II in acid solutions was described bluish red. The bluish tinge becomes more
by Atack (1), who emphasized that these com- noticeable with increasing PH; i.e., the salts of
pounds are salts, not complexes. alizarin red S formed in strongly alkaline solu-
Formation of insoluble or poorly soluble tions resemble those of alizarin. In the weakly
calcium or barium salts of sulfonated dyes is alkaline region (around pH 9) the two types of
widely used in industry in the preparation of reactions probably overlap. This assumption
pigments; these dye salts are referred to as lakes is supported by observations that at pH 9
or toners. In the dye and pigment industry calcium deposits are stained selectively by acid
the term lake denotes a poorly soluble or insolu- dyes for lakes and by alizarin and alizarin blue
ble salt of a water-soluble acid or basic dye (31, S. In analogy to the formula for the calcium-
38, 41, 48). Sulfonated dyes suitable for this aluminum compound of alizarin red S given by
purpose are classified as “acid dyes for lakes” Baumann and Hensel (4), the calcium salt
(10). In the precise terminology of dye chemistry formed under alkaline conditions would be (Ca-
the designation “acid dye” signifies that these ali.zarin-3-SO3)2Ca. As in the case of alizarin,
dyes do not contain chelating groups. Dyes the color change of alizarin red S cannot be
capable of chelate formation with metals are regarded as evidence for complex (chelate)
classified as mordant dyes. Of course, certain formation. Striking color changes upon salt
mordant dyes can also form lakes, i.e. the readily formation with calcium or barium have also
soluble sodium or potassium salts can be con- been observed with acid dyes for lakes which
verted into poorly soluble calcium or barium do not contain chelating groups (31).
salts. Barium salts (lakes) of sulfonated anthra- The use of the term lake as a synonym for
quinone dyes have been described by Mattiello chelates apparently caused some ambiguity
(31). Alizarin red S is now mainly used in the even in the chemical literature. For example,
preparation of lakes; in textile dyeing it has been compounds of alizarin red S with hafnium and
largely replaced by other dyes (4). other cations of the titan group have been in-
The effect of pH on lake formation by acid terpreted as chelates (innere Kompl&xsalze) (12),
dyes has been discussed by Pratt (38). In these yet Venkataraman (48) cited spectroscopic
chemical studies an excess of metal was used. evidence that the hafniurn-alizarin compound
At pH 5.5 and below, all dye molecules reacted is a salt, not a chelate. Hence, reports concerning
with the metal. Lake (salt) formation decreased complex (chelate) formation of alizarin and its
around pH ; in alkaline solutions only a mod- derivatives with various cations should be
erate amount of dye was bound by the metal. interpreted with caution. The investigations
This pH effect is very inconvenient for histology. by Kiel and Heertjes (25-28) indicate that
At pH 5.5 and below, basic groups of tissues also infrared spectroscopy is at present the most
react with sulfonic acid groups of dyes; this reliable method for distinction between salts
diffuse coloration of sections made identification and chelates of alizarin and its derivatives.
of small calcium deposits very difficult. The Alizarin red S is as nonspecific for calcium as
choice of acid dyes for lakes is therefore limited the parent dye alizarin. These observations art
to those dyes which produce strong selective in agreement with findings by Dahl (11). Im
coloration of calcium deposits in the nonoptimal addition to the compounds listed in Table I
pH range 7-9. alizarin red S is also very sensitive towart
In contrast to acid dyes for lakes, hydroxy- the ions of uranium, titanium, bismuth ank
anthraquinone dyes containing acid groups can thallium (15), and toward zirconium, hafnium
readily form salts over a wide p11 range. In and thorium (12). The differences in color
122 PUCHTLER, MELOAN AND TERRY

among various alizarin red S-metal compounds solutions differ significantly from batch to batch
tested were insufficient for identification of and apparently depend on the impurities in the
the cations in a blind study. As already men- dye samples. Of the five samples tested in this
tioned, the same difficulty was encountered with study, only two produced passable selective
alizarin. staining of calcium deposits at pH values below
Binding of acid dyes for lakes by calcium 7. The yellow impurities of the other three dye
deposits in tissues: The selective coloration samples competed effectively with alizarin red
of calcium deposits by several acid dyes for S for binding sites in calcium deposits. Thus the
lakes shows that salt formation between sul- discrepancies between recommended pH values
fonic acid groups and calcium or other cations and reported difficulties in reproducing the work
occurs under the conditions of histologic tech- of others can readily be explained by differences
nique. The abolition of staining by pretreatment in the dye samples used by various workers. The
of sections with dilute acetic acid substantiates low pH values of aqueous solutions of the impure
the conclusion that these dyes react with cal- dyes and the staining properties of the con-
cium or other metallic cations and not with taminants indicate that these compounds are
basic groups of proteins. Preliminary compara- strongly acid. Since many commercial samples of
tive studies of acid dyes for lakes and other azo dyes contain intermediates (44), it seemed
acid dyes indicate relations between the configura- conceivable that batches of alizarin red S may
tion of dye molecules and selectivity for calcium also be contaminated with sulfonated by-
deposits. Even dyes which differ only in the products of the manufacturing process. According
position of one sulfonic acid group can show to Fierz-David and Blangey (13) and Venkatara-
strikingly different staining properties. For man (48), 2-anthraquinonesulfonic acid is used as
example, the monoazo dye Bordeaux red B starting material in commercial production of
(1-naphthylamine -‘ 2-naphthol-3 , 6-disulfonic alizarin. A concentrated solution of 2-anthra-
acid) colored calcium deposits strongly and quinonesulfonic acid, NaOH and NaC1O3 or
selectively, but Ponceau 6R (1-naphthylamine NaNO3 is heated at 185#{176}C
and 5-6 atm pressure
2-naphthol-6 ,8-disulfonic acid) showed no affinity for 48-72 hr. The 2-anthraquinonesulfonic acid
for calcium deposits. Further investigations of is converted into 1-hydroxyantb.raquinone-2-
the relations between dye structure and affinity sulfonic acid; in a second step the disodium salt of
for calcium are in progress. Previous studies alizarin is formed. Completion of the reaction is
showed relations between fluorescence and indicated by loss of fluorescence; 2-anthra-
structure of azo dyes (39). These relations were quinonesulfonic acid and 1-hydroxyanthra-
found to apply also to the calcium lakes. Of the quinone-2-sulfonic acid are strongly fluorescent,
dyes tested, Ponceau 2R was found most suitable but alizarin is nonfluorescent (13). Alizarin red S
for fluorescence microscopic studies of calcium is prepared by sulfonation of alizarin. When
deposits. tissue sections stained with impure samples of
The effects of different fixatives and of varia- alizarin red S were viewed in ultraviolet blue
tions in fixation and embedding procedures on the light, the structures colored by the yellow
binding of acid dyes for lakes and the specificity contaminants showed moderate to strong yellow-
of these dyes for various cations have not yet ish red fluorescence. Calcium deposits stained red
been determined. These dyes were included in this by alizarin red S were nonfluorescent. These
study for the sole purpose of obtaining informa- observations suggest that the yellow impurities
tion concerning the role of the sulfonic acid group consist, at least in part, of intermediates.
of alizarin red S in the staining of calcium deposits Further disadvantages of acid solutions of
in alcohol- or Carnoy-fixed tissues. Therefore, alizarin red S are diffusion artifacts and loss of
until further data become available, these dyes calcium. Gomori (19) recommended buffer solu-
should not be regarded as substitutes for alizarin tions of pH 4.5-5 for removal of calcifications in
and its derivatives. tissues. It is therefore not surprising that solutions
Binding of alizarin red S by tissue sections: of alizarin red S at pH 4.8 or below removed
The recommended pH values for solutions of significant amounts of calcium, and diffusion
alizarin red S vary from 4.1-4.2 (32) to 6.3-6.5 artifacts became evident within 1 mm. Hence,
(11). Our experiences indicate that the staining acid solutions of alizarin red S are unsuitable for
patterns obtained with alizarin red S in acid studies of the localization of calcium deposits at
 ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM 123

the intracellular level, e.g., in muscle fibers or sections with 10% unbuffered formaliti for 15 hr
renal epithelial cells, and for semiquantitative removed all calcium deposits. Similar treatment
estimates. In the discussion of methods for vith Zenker-formol (Spuler, Iaxiiuow) was
enzymes, Gomori (19) stated explicitly that equally effective. Ilence, the major loss of
calcium salts cannot be used effectively below pH calcium seems to occur (luring fixation and not
8.5 because of their solubility and consequent at later stages of the embeddmg procedure.
diffusion artifacts. Such diffusion artifacts were According to 1)ahl (11), solutions of formaliti
very obvious in sections stained with alizarin red buffered to I)11 7.2 or 8.35-8.7 also caused
S at pH 7.2, but were absent in sections stained significant loss of calcium salts and were clearly
with alizarin red S at pH 9, the pH value recom- inferior to al)solute alcohol. Obviously, these
mended by Gomori (19) for procedures employing fixatives are unsuitable for histochemical studies
calcium salts. Parenthetically, alkaline solutions of calcium deposits. That (‘arnoy’s fluid, which
of alizarin red S were already in use around the contaim 10% acetic acid, preserves calciuni
turn of the century. Roehl (43) recommended deposits as wellas absolute alcohol is probably due
differentiation in alkaline media or addition of to the very low dissociation of acetic acid and pOOr

NaOH, Li2CO3 or NH3 to aqueous solutions of solubility of calcium salts in absolute alcohol and
alizarin red S. Differentiation is unacceptable in chloroform.
histochemistry (34) and impractical for general
hospital pathology. In this study, 0.5% solutions REFEHENCES
of alizarin red S buffered to approximately pH 9 I. Atack, F. W.: A new reagent for the detection
and colorimetric estimation of aluminum.
yielded optimal coloration of calcium deposits. As
J. Soc. Chem. md. 34: 936, 1915.
already mentioned, at this pH the yellow im- 2. Attree, G. F. and Perkin, A. G.: Reduction
purities present in three dye batches were no products of the hydroxyanthraquinones.
longer bound by tissue sections; the staining J. Chem. Soc. 134: 144, 1931.
3. Barka, T. and Anderson, P. J.: Histochemistry:
patterns obtained with the five dye batches Theory, Practice and Bibliography. Hoeber
tested were indistinguishable. Higher p1-1 values Medical Division, Harper and Row, Pub-
ushers, Inc., New York, 1963.
were found impractical because the sections 4. Baumann, 11. and ilensel, II. H.: Neue Metal!-
tended to become detached from the slides. komplexfarbstoffe, Struktur und farberische
Eigenschaften. For(seh r. Chc?n. Forsch. 7:
Because solutions of alizarin red S buffered to
643, 1967.
pH 9 do not cause noticeable diffusion artifacts 5. Becher, S.: Untersuchungen liber Echtfarbung
or loss of calcium salts, staining can be prolonged der Zeilkerne mit k#{252}n.stlichen Beizenfarb-
stoffen und die Theorie des hislologischen
to 1 hr. Intensity of coloration of calcium,
Farbeprozesses mit gelosten Lacken. Verlag
aluminum or other salts by alizarin red S in- von Gebruder Borntraeger, Berlin, 1921.
creases with the time allowed for lake (salt) 6. Caley, E. II.: The Leyden Papyrus X. An
English translatioii with brief notes. .1.
formation (49). Since replacement of anions in Chem. Ed. 3: 1149, 1926.
calcium deposits by the dye progresses gradually 7. Caley, E. R.: The Stockholm Papyrus. An
from the periphery, staining for 30-60 mm English translation with brief notes. .1.
Chem. Ed. 4: 979, 1927.
improved coloration of crystalline areas more 8. Cameron, U. H.: The staining of calcium.
than 10 in diameter. The slight grayish pink J. Path. Bact. 33: 931, 1930.
9. Colour In(iex, Ed. 2, Vol. 1. Society of 1)yers
background stain did not interfere with the study
and Colourists and American Association
of small intracellular calcium deposits and of Textile Chemists and Colorists, Lowell,
actually served as a counterstain. As pointed out Mass., 1956.
10. Colour Index, Ed. 2, Vol. 2. Society of 1)yers
by Barka and Anderson (3), counterstaining is
and Colourists and American Association
undesirable from a histochemical standpoint. ofTextile Chemists and Colorists, Lowell,
Because calcium and other salts of alizarin red S Mass., 1957.
11. 1)ahl, L. K.: A simple and sensitive histo-
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