On the History and Mechanism of Alizarin and Alizarin Red s Stains

http://jhc.sagepub.com/Journal of Histochemistry & Cytochemistry

http://jhc.sagepub.com/content/17/2/110The online version of this article can be found at:

DOI: 10.1177/17.2.110

1969 17: 110J Histochem CytochemHOLDE PUCHTLER, SUSAN N. MELOAN and MARY S. TERRY

ON THE HISTORY AND MECHANISM OF ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM

Published by:

http://www.sagepublications.com

On behalf of:

Official Journal of The Histochemical Society

can be found at:Journal of Histochemistry & CytochemistryAdditional services and information for

http://jhc.sagepub.com/cgi/alertsEmail Alerts:

http://jhc.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

What is This?

- Feb 1, 1969Version of Record >>

at CHRISTIAN UNIV on December 18, 2013jhc.sagepub.comDownloaded from at CHRISTIAN UNIV on December 18, 2013jhc.sagepub.comDownloaded from

http://jhc.sagepub.com/


http://jhc.sagepub.com/content/17/2/110

http://jhc.sagepub.com/content/17/2/110



http://www.histochemicalsociety.org/

http://www.histochemicalsociety.org/

http://jhc.sagepub.com/cgi/alerts

http://jhc.sagepub.com/cgi/alerts

http://jhc.sagepub.com/subscriptions

http://jhc.sagepub.com/subscriptions

http://www.sagepub.com/journalsReprints.nav

http://www.sagepub.com/journalsReprints.nav

http://www.sagepub.com/journalsPermissions.nav

http://www.sagepub.com/journalsPermissions.nav

http://jhc.sagepub.com/content/17/2/110.full.pdf

http://jhc.sagepub.com/content/17/2/110.full.pdf

http://online.sagepub.com/site/sphelp/vorhelp.xhtml

http://online.sagepub.com/site/sphelp/vorhelp.xhtml





110

THE JOURNAL OF HI8TOCHEMISTRY AND CYTOCHEMI8TRY

Copyright © 1969 by The Histochemical Society, Inc.

Vol. 17, No. 2

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, notchelates. In dye chemistry the term lake denotes a poorly soluble or insoluble salt of awater-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. Aciddyes 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 coloredcalcium deposits selectively around pH 9; neutral and acid dye solutions produced severediffusion artifacts. Chemical data indicate that alizarin red S can react with calcium via itssulfonic acid and/or its OH groups.

In histology alizarin red S and von Kossa’s

technique are commonly used for demonstration

of calcium salts. Von Kossa’s procedure is

believed to visualize phosphate and carbonate

anions, whereas alizarin red S reacts with calcium

and other cations. Both methods have been used

for diagnosis of calcium deposits. However,

during investigations of early arteriosclerotic

lesions we observed striking discrepancies be-

tween the amounts of “calcium” demonstrated

by alizarin red S and by von Kossa’s technique.

For example, in some arteries von Kossa’s

procedure colored segments of the internal

elastic membrane black, yet adjacent sections

utterly refused to bind alizarin red 5, and vice

versa. Obviously, for an understanding of the

processes occurring in these arteriosclerotic

lesions, it was essential to obtain information

concerning the histochemical significance of these

procedures. A preliminary perusal of the literature

showed differences between chemical and histo-

logic concepts; e.g. chemists classify the calcium-

alizarin or alizarin red S compounds as a salt,

whereas in histology and histoehemistry it has

‘Dedicated with admiration to Dr. R. D. Lillie,who by his far ranging knowledge of the historyand chemistry of dyes, elevated staining from acraft to a branch of histochemistry.

2 This investigation was supported by a grant-in-aid from the Georgia Heart Association andUnited States Public Health Service ResearchGrant HE 12147 from the National Heart Insti-tute.

been referred to as a lake or complex. Further-

more, since the publication of the reviews by

Harms (22) and McGee-Russell (32), new

chemical and infrared spectroscopic data on

calcium-alizarin-alizarin red S and related com-

pounds have become available. It was therefore

deemed of interest to reinvestigate the mechanism

of alizarin and alizarin red S stains for calciumand to search for a method which would avoid

diffusion artifacts. Since this project was part of

a study of early arteriosclerotic lesions, investiga-

tions were limited to calcifications in soft tissues;

bone and teeth were excluded. These studies and

a review of pertinent literature are presented in

this report. Observations on the mechanism of

von Kossa’s procedure will be published sepa-

rately.

MATERIALS AND METHODS

Experiments were carried out on human autopsymaterial. Blocks of tissues were fixed in absolute

alcohol, Carnoy’s fluid (absolute alcohol-chloro-form-glacial acetic acid, 6:3:1), 10% unbuffered

formalin or Zenker-formol (Spuler, Maximow)and embedded in Paraplast by Autotechnicon.Sections were cut at 5 p.

One batch of alizarin siccum (Chroma, C.I.58000), four batches of the biologic stain alizarinred S (Chroma, C.I. 58005), one sample of thecorresponding textile dye Diamond red W (VeronaDyestuffs) and one sample of alizarin blue S

(Chroma, C.I. 67415) were used. Solutions, 0.1and 0.5%, of the sulfonated dyes in distilled water

at CHRISTIAN UNIV on December 18, 2013jhc.sagepub.comDownloaded from



ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM 111

and in 0.5% aqueous solutions of Brook’s buffers,pH 4.8, 7.2 and 9.0 were prepared. To determine

possible effects of the buffer salts on the stainingreaction, Sorensen’s phosphate buffer and barbital

buffer, pH 7.2 and 9, were substituted for Brook’sbuffer. Owing to the poor solubility of alizarin,

only saturated solutions of this dye in pH 7.2 and9 buffers and in 0.3 and 1% aqueous NaOH were

tested. Sections were stained for 5 mm or 1 hr,rinsed in buffer solution for about 5 sec, dehy-drated in three changes of absolute alcohol,cleared in xylene and mounted in Permount. Other

series were stained with alizarin red S as recom-mended by McGee-Russell (32).

To obtain information concerning the selec-

tivity of alizarin and alizarin red S for calcium,the reactivity of various salts was studied in

model experiments. To avoid lengthy repetition,

the compounds used are itemized only in Table I.Approximately 500 mg of the substance (A.C.S.reagent grade) to be studied were placed near

one end of a slide. Two to 3 drops of the dye solu-tion were added; another drop was placed at the

other end of the slide to serve as a color standard.The interaction between test substance and dye

solution was observed between parallel andcrossed polaroids for 2-5 mm or until no furtherchanges occurred. This procedure is a modification

of the technic recommended by McGee-Russell(32) for staining of calcium in tissue sections.

In another series the salts were placed on slides,

mixed with gelatin, air-dried, fixed in formalde-hyde vapor or Carnoy’s solution and stained as

described above for tissue sections.To investigate the role of the sulfonic acid

group of alizarin red S in the staining of calciumdeposits in tissues, the staining patterns of sal-fonated dyes without chelating groups recom-

mended by Pratt (38) or the Colour Index (10)for preparation of barium or calcium lakes were

studied. The following dyes were employed:Bordeaux red B (C.I. 16180), brilliant black (C.I.27260), croceine orange Y (C.I. 15970), fast light

rubine BL (C.I. 17065), fast red S (C.I. 15620) and

Ponceau 2R (C.I. 16150). To determine whether

or not high affinity for earth alkalis is a peculiarityof certain acid dyes, a series of sulfonated dyes(without chelating groups) was selected for com-parison, namely Acilan cosine E (C.I. 14710),Acilan red S2B (C.I. 23910), amaranth (C.I.16185), azophloxin GA and fast crimson GR (C.I.

18050), azorubin S (0.1. 14720), Benzo fast scarlet4BS (C.I. 29160), Benzo fast scarlet 4GS (C.I.29185), Biebrich scarlet WS (0.1. 26905), brilliant

scarlet 6R (C.I. 16255), croceine scarlet 6R (C.I.16255), croceine scarlet MOOP (0.1. 27290), di-phenyl green GPD (C.I. 30295), durol black 2B

(0.1. 26370), fast wool red GL (C.I. 17045), levanol

fast scarlet FGN (0.1. 18020), levanol yellow 6G(C.I. 23900), Niagara sky blue 6B (0.1. 24410),

orange G and wool orange 2G (0.1. 16230), Siriussupra red 4BLA (0.1. 29065), Sirius supra tur-quoise LG (0.1. 74180), Solophenyl orange TGL

(0.1. 40215 and 40220) and tartrazine 0 (0.1.19140). Sections were stained in 0.5% solutions

of these dyes for 5 mm or 1 hr.The dye names given above are the trade names

used by the manufacturers. Previous paper chro-matographic studies showed significant discrep-ancies between the composition of some dye

samples carrying the same Colour Index numberobtained from different manufacturers (44). Itwas therefore deemed expedient to use the trade

names of the dyes employed rather than syno-nyms. Other brands of these dyes may or may notyield similar staining patterns.

Calcium and related salts were removed fromsections by treatment with 5% aqueous acetic

acid for 5 or 10 mm. Sections were then washedin distilled water and stained as described above.Since alizarin-metal chelates are insoluble inacetic acid, whereas salts of alizarin are readily

soluble (22), stained sections were treated with5% aqueous acetic acid for 5 mm to obtain in-

formation concerning the nature of the bondbetween dyes and calcium deposits in tissues.

Paper chromatograms of the four batches of

alizarin red S and of Diamond red W were pre-

pared according to the method described byRosenthal, Puchtler and Sweat (44).

A Reichert Zetopan microscope equipped with

a twin lamp unit (tungsten and mercury vapor

lamp), bright field and dark field condensers, was

used for comparison of staining and fluorescencemicroscopic patterns. The combination of ultra-violet blue pass filter BG 12/3 mm and ultravioletblue barrier filter Sp. 3 (GG 9/1 mm + OG 1/1.5mm) was employed. Dyes which were not fluores-cent under these conditions were studied also

with ultraviolet pass filter UG 1/1.5 mm andbarrier filter Sp. 2 (GG 13/1 + 3 mm + Wratten

foil 2B).The term “calcium deposits” is used in a de-

scriptive sense without strict chemical denota-tions; these areas may or may not contain othercations in addition to calcium.

RESULTS

Effect of fixatives: Comparison of sections

from the same organ fixed in the solutions listedabove and stained under identical conditionsshowed striking differences. Coloration of calciumdeposits was most intense in alcohol- and Carnoy-fixed sections; no difference was observed between

these two fixatives. In formalin- and Zenker-




112 PUCHTLER, MELOAN AND TERRY

fixed material calcium deposits were weakly to

moderately colored. Staining of calcium deposits

decreased with the duration of storage in formalin

and was abolished after 2-3 weeks. The stainingproperties of material fixed in Zenker-formol

varied significantly and seemed to depend on theduration of fixation and subsequent washing.Therefore, unless otherwise stated, all descriptionsand comments hereafter refer only to alcohol- and

Carnoy-fixed tissues.Binding of alizarin: Alizarin was practically

insoluble in buffers of pH 7.2 and did not staincalcium deposits. Saturated solutions of alizarin

in buffers of pH 9 or in 0.3% aqueous NaOHcolored most calcium deposits moderately; very

small deposits were only faintly stained. Veryintense bluish red to purple coloration of all

calcium deposits within 5 mm was obtained with

saturated solutions of alizarin in 1% aqueous

NaOH; other tissue structures remained un-stained. Small intracellular deposits were clearlydelimited and there were no diffusion artifacts.Sections stained with this procedure were used as

standards in assessing the specificity and intensityof coloration obtained with other staining tech-niques described below. Unfortunately, in this

strongly alkaline solution sections tend to becomedetached from slides and the method is thereforeinconvenient for general use in hospital pathology.

Attempts to substitute 1% NaOH in 70% ethanol

as a dye solvent were unsuccessful; under theseconditions calcium deposits were only weakly to

moderately colored, even when staining wasprolonged up to 1 hr.

Aqueous unbuffered solutions of alizarinred S: The staining properties of 0.1% solutions of

alizarin red S varied widely from batch to batch.One batch, which had been purchased severalyears before, yielded deep orange-red coloration ofcalcium deposits within 5 mm; other tissue

structures were stained light pink. When stainingtime was extended to 1 hr, the intensity of colora-tion of calcium deposits was strikingly decreased

and other tissue structures were moderately

stained. This loss of contrast made it difficult toidentify small foci of calcification. Practically

identical staining patterns were obtained with thecorresponding textile dye Diamond red W. A

sample of alizarin red S bought in the course ofthis study stained calcium deposits yellowishorange; all other tissue structures were coloredbright yellow (staining time 5 mm). Two further

samples from different batches of alizarin red Syielded similar staining patterns. No trace of thered dye could be found in sections stained for 1hr in the three dye samples containing yellowimpurities; tissues were colored uniformly yellow.

With all dye samples tested, diffusion artifacts

around calcium deposits were already severe in

sections stained for 5 mm. Individual granules,

e.g., in muscle fibers or renal epithelial cells, were

not recognizable; the cells were colored uniformlyorange-red.

Alizarin red S in buffer solutions, pH 4.8:

The staining patterns obtained with these solu-tions were very similar to those produced byunbuffered aqueous solutions.

Paper chromatograms of the old batch of alizarinred S and of Diamond red W were very similar.

The dye moved in a well defined band which wasframed by traces of bluish pink material. Theother three dye samples contained less alizarinred S and showed significant amounts of yellow

impurities which moved more slowly than thedye; the dye band was not homogeneous butincluded a brownish substance. The pH values of0.1% aqueous solutions of these dyes were: old

batch of alizarin red S, 3.5; Diamond red W, 3.7;other batches of alizarin red 5, 2.7, 2.9 and 2.9;

thus the impurities were apparently more acidthan alizarin red S.

McGee-Russell’s procedure: Under the condi-tions of McGee-Russell’s procedure (32) all dye

samples stained calcium deposits deep orange.Intensity and hue of background coloration varied

with the amount of impurities in the dye samples,as described above for 0.1% aqueous solutions ofthese dyes. When staining time was limited to 1

mm, diffusion of the orange substance was moder-ate. However, small intra- or extracellular gran-

ules, which stood out clearly in adjacent sectionsstained with alkaline solutions of alizarin, alizarinred S or alizarin blue S (see below), could not beidentified; such areas were colored uniformly

orange.Alizarin red S in buffer solutions, pH 7.2:

Calcium deposits were colored orange-red. Sec-tions stained for 5 mm showed moderate diffusion;granules of calcified material in cells and muscle

fibers were not distinguishable. All other tissue

structures were stained faintly pink; binding ofthe yellow impurities in three dye batches was

abolished. However, some yellow colorationpersisted in and around calcium deposits, particu-larly between closely spaced lesions. In sections

stained for 1 hr the intensity of coloration ofcalcium deposits and background staining weremoderately increased, and diffusion aroundcalcified areas was more marked.

Alizarin red S in buffer solutions, pH 9: In

sections stained for 5 mm calcium deposits werestained red without a yellow tinge; all other

structures were unstained or were colored faintlypink. Even small granules of calcified material

stood out clearly against the practically colorlessbackground. No trace of yellow could be found in





these sections; the staining patterns obtainedwith the different dye batches were identical.Intensity of coloration of calcium deposits in-creased with time of staining up to 1 hr. No halosor other signs of diffusion were found in this

series; even in sections stained for 1 hr calciumdeposits were sharply delineated, e.g., in renalepithelial cells or cardiac muscle fibers containingnumerous granules.

Alizarin blue S: Solutions of this dye indistilled water or in pH 7.2 buffers stained fairlylarge calcium deposits weakly; small deposits were

barely recognizable. Calcium deposits were

moderately colored after staining for 5 mm in a

0.5% solution of alizarin blue S in pH 9 buffers;

optimal coloration of calcium deposits wereobtained by staining for 1 hr. Under this conditioncalcium deposits were colored deep blue to reddish

blue and were sharply delimited. All other tissuestructures remained unstained.

Acid dyes for lakes: In sections stained for 5mm, the sulfonated azo dyes Bordeaux red B,brilliant black, croceine orange Y, fast lightrubine BL and Ponceau 2R stained calcium de-

posits selectively. Intensity of coloration wasincreased when staining time was prolonged to 1hr. Diffusion did not occur; calcium deposits were

sharply delineated. Excellent contrast betweenintensely colored calcium deposits and practically

unstained tissues was obtained with Bordeaux

red B, brilliant black and fast light rubine BL.The deep yellow coloration imparted by croceineorange Y and the yellowish red color of Ponceau2R were found less convenient for studies of

fine granules in early stages of calcification, e.g.,

in arteriosclerosis and in renal tubular epithelial

tells. The pH value of the 0.5% aqueous solutionsof these dyes ranged from 7.0 (brilliant black) to

9.7 (Bordeaux red B). Solutions of these dyes

buffered to pH 4.8-5 stained all tissues stronglyand identification of calcium deposits became

difficult or impossible.Aqueous 0.5% solutions of azorubin S (pH 6.5),

Biebrich scarlet WS (pH 6.2), fast red S (pH 6.2)and croceine scarlet MOOP (pH 9) stained calciumdeposits intensely; other tissue structures wereweakly or moderately colored. The stainingpatterns produced by these dyes closely resembled

those obtained with aqueous solutions of alizarinred S at pH 5 or below. Contrast between calcium

deposits and tissues decreased with prolongationof the staining time beyond 5 mm, owing toincreasing background staining. The other sal-

fonated dyes tested colored tissues and calciumdeposits more or less uniformly within 5 mm.

Selectivity of sulfonated dyes for calcium depositswas independent of the pH of the dye solution;the pH values of 0.5% aqueous solutions of non-

selective dyes varied from 6.2 to 9.5 and werecomparable to those of acid dyes for lakes.

In sections stained with Ponceau 2R the strongred fluorescence of calcium deposits contrastedwell with the faint greenish gray primary fluores-cence of other tissue structures. Calcium depositsstained with Bordeaux red B were weakly tomoderately fluorescent. Sections treated withbrilliant black, croceine orange Y or fast lightrubine BL did not show secondary fluorescence.Dyes which produced background staining were

unsuitable for fluorescence microscopy. For

example, azorubine 5, which produced slight tomoderate background staining, rendered calciumdeposits intensely fluorescent, but other tissue

structures showed strong secondary fluorescence.Treatment of sections with acetic acid: No

calcium deposits could be found in sections pre-

treated with 5% aqueous acetic acid for 5 or 10mm and stained with alizarin, alizarin red S oracid dyes for lakes; other tissue structures werefaintly colored or remained unstained. When

stained sections, which showed intensely colored

calcium deposits, were treated with 5% aqueousacetic acid for 5 mm, this coloration was removed.Occasionally a few large calcified areas remainedfaintly stained.

Model experiments: Dispersions of various

calcium and other salts in gelatin were foundunsuitable. When such model slides were stainedin acid dye solutions, the fairly acidophilic gelatinalso bound the dye and evaluation of the reactions

of embedded salts became difficult, if not im-possible. Alkaline dye solutions (pH 9-12) causedswelling of the gelatin, and membranes of gelatintended to become detached for the slides. These

series were therefore regarded as unsuitable forcritical evaluation.

In contrast, when the dye solutions were addedto salts on a glass slide under microscopic control,

changes of color were easily observable. Theresults of this series are summarized in Table I.

Obviously, alizarin and alizarin red S were notspecific for calcium, nor for earth alkalis, butreacted with a wide variety of cations. Further-

more, the colors of the dye salts were also notspecific for certain cations. The formation ofcolored dye salts depended, at least in part, on

the solubility of the compounds tested. Forexample, the practically insoluble BaSO4 did notreact with the dye solutions; in contrast, thesoluble BaCh produced significant color changes.

The color changes of the dyes commenced at thesurface of the crystals. As the crystals dissolved,

the metal-dye compounds diffused into the sur-rounding solution. A strongly colored layer wasformed on the surface of poorly soluble crystals,

but the interior of the crystals remained unstained




TABLE I

Staining of Various Salts by Alizarin Red S and Alizarina

SaltAlizarin Red

S,pH4.8Alizarin Red

S,pH9Alizarin 1% NaOH,

pHll.8

CuCl dR dR h R h, dOll-BR dP-RVCuCl2�2H2O P h BP YR-BR decolCuSO4�5H2O dRV dR RV VCuSO4(NH4)zSO4�6H:O PV, B PV, B PV, B sl PV, BCuCl2�2NH4Cl RV RV RV RMgSO4�7H2O - dR dR dBR

MgCO3.Mg(OH),.nH,O dRsR dIlsR dR dRsRNH4Cl�MgCl2 - dRaR dRsR dR.sR

MgCl2�6H2O - dRsR dRsR dRaR, dBRCaCl,�2H2O P dBR BrR RCaCO, YR BR sl R dVR

CaC2O4�H2O YR, R p dR BrR dBRVCaSO4�2H2O YR p, h dV BR VCa(H,P04)2.H,O Y h R h Y YCaHPO4 Rh dBR BrR VCaio(OH)2(P04)6 YR dR BR dV, BrSrCO3 Ph - - -

SrCl2�6H,O OR dR p BrY RB pBaCl2�2H,O YP dRsR YO dV pBaSO4 - - - -

ZnCO, R-P si 0 - PBrZnC1, P p dR dR YR

CdC1, - dR dR RV p, 0-BR

3CdSO4�8H2O - dR dR V h

HgCl: - BrY - dBrHgSO4 - Lighter 1Y Y

Al,(SO4),.18H20 dY OR OR p ORAl2(S04),.18H,O OR V-OR Y, 0 BR p, 0CaSO4�2H20SnCI,#{149}2H,0 R p R p R p B-ORSnCl4�5H20 Y Y Y YPbCO1 YRh - - BRpPbC12 OR h dR BrV Br pBiCl, Br Br Y YMnCl2�4H2O dY dR dBR BrRMnSO4.H20 - P, BR p 0-BR BR pFeCl2�4H20 B p P-Y B, R, Y YFeCl, - slBR,Y - YFeNH4(SO4)2� 12H2O BrV p si R RV h RFe,(S04), (about 6H20) - BrV BrV h R hFeCl,�2NH4Cl�H,O - - BrV R-BrFeSO4.7H20 RV RV V V p

Fe(NH4)2(S04)2�6HzO BrV dR RV p V pCoOl, 0 dBR d.BR R, YEt, V, GCoSOg�7H,0 - dBR dBR-V R hNiCl,�6H,O P R, B R, B BR pNi(C,H,O,),.4H,O dBR dBR BR BPCr(NH4)(S04)2.12H,O RO-dR BrR BrR h Gy pCrCls�6H,O[Cr(H,O)6JCl, Br p dBr p Br p Br pCr(C,H,0,),.H,0 dR dR h Br h V pK,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





or retained the original color. Thus the dye solu-tions apparently did not penetrate the crystals,

but reacted only with the superficial layers.

Frequently, such salts resembled some calcium

deposits in tissues which also showed an intenselycolored periphery and weakly colored or unstained

center. The progressive breakdown of crystal

structure and simultaneous formation of coloredcompounds could easily be followed by alternate

observation between crossed and parallel polar-

oids. A few compounds did not dissolve duringthe period of observation; only a thin colored

layer was formed on their surface. This group

included CaC,04, SrCO, and PbCO,.A few compounds, e.g., MgCl,, did not react

with alizarmn red S at pH 4.8, but produced color

changes with the other dye solutions tested.

Ca(H,P04), reacted slightly with alizarin red S

in pH 9 buffer solution but not with alizarin redS in pH 4.8 buffer solution or with alizarin. Sinceit appeared possible that this phenomenon might

be due to a lowering of the pH of the dye solutionby the dissolving salt (see “Discussion” below),2-3 drops of the dye solvents were added to crys-tals of Ca(H,,P04), and the approximate pH of

the resulting saturated solution was determined

by indicator papers. The pH values were 2-2.5.

DISCUSSION

The literature on alizarin and alizarin red S

stains for calcium deposits in tissues has been

reviewed comprehensively by Harms (22) and

McGee-Russell (32). These histologic procedures

were originally based on ancient traditions in

textile dyeing with madder (18). The term “lake”

for the alizarin-calcium compound was ap-parently derived from half-forgotten early chemi-

cal concepts. It therefore seems appropriate to

review briefly the history of dyeing with madder

and alizarin. Explanations of the chemical

mechanism of ali.zarin and alizarin red S stains,

whether the term lake implies complex or salt

formation, are scanty. This discussion therefore

is limited mainly to chemical aspects of theinteraction of these dyes with calcium and other

cations.

History of madder dyeing: Madder was

used for dyeing of textiles in early antiquity.

According to Forbes (14), the oldest samples are

pieces of cotton from Mohenjo-Daro dating

back to the third mifiennium B.C.; in Egypt,

textiles dyed with madder were found in tombs

of the period of the XVIII-XXth Dynasties.

The oldest extant samples of recipe books for

dyeing are the Leyden Papyrus X and the

Stockholm Papyrus, which are believed to have

been written about the end of the 3rd century

A.D. (6). However, some of these recipes refer

to originals of 300 B.C. at the latest and probably

much earlier (14). The Stockholm Papyrus (7)

contains numerous mordanting procedures and

recipes for dyeing with madder and other natural

dyes. Lime water and alum dissolved in vinegar

(aluminum acetate?) are frequently recom-

mended as mordants. For dyeing a rose color,

wool was smeared with ashes, washed in liquid

from potter’s clay and mordanted; after being

rinsed in salt water, the wool was dyed in a

solution of madder, bean meal and white oil in

rain water, and aftertreated with alum. To

obtain a purple coloration, textiles were treated

first with a blue dye and then mordanted and

dyed with madder as described above, except

that the concentration of madder was doubled.

However, it was already known that the shade

of madder dyeings could be varied by the use of

different mordants (14). Postmordanting with

alum was recommended to prevent fading; this

procedure seems to be a precursor of the after-

chroming or coppering procedures of modern

textile industry to improve fastness properties.

According to Caley (7), the methods described

in these papyri were essentially the ones used

during the next 1500 years until the advent of

synthetic dyes.

Parenthetically, the dyeing of wool on live

animals, commented upon so disapprovingly by

Pliny, is mentioned in the Stockholm Papyrus,

which gives “Book 3 of Africanus” as reference.

The animals were washed and areas to be dyed

were mordanted with a solution of alum in

vinegar. According to Pliny (23), the dyes were

applied “by the means of certain barks of a foot

and a half long dipped in these colours, and so

imprinted and set upon their fleeces as if riotous

wantonness and superfluitie should force Nature’sworke, and make wool grow of the colour.”

However, it seems much more probable that

these decorations were simply proprietary im-

prints; dyes are still used for this purpose in the

Basque region when the sheep are taken to the

mountains for summer grazing. Since alizarin on

an aluminum mordant has light fastness 7-8 (9),

it seems well suited to withstand fading on

animals exposed to the strong sunlight of Egypt.

In contrast to branding, this ancient procedure





did not interfere with later conversion of the

skins to leather.

Madder was widely distributed in the Old

World and cultivated near Rome in classical

antiquity (46). The history of madder dyeing in

Europe has been reviewed by H#{252}bner (23) and

Hailer (21). According to H#{252}bner (23), the role

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

the Colour.” During the following centuries

madder dyeing apparently came under the

influence of alchemy. To “animalize” textiles,

repeated treatments with cow, sheep or goat

dung prior to mordanting and dyeing and an

aftertreatment with dung were considered es-

sential by Bancroft in 1813 (23). These alchemical

practices were challenged less than a century ago

by Schlieper and Baum, who recommended

mordanting wite aluminum and calcium salts and

dyeing in a solution of alizarin and calcium salts

(21), a procedure similar to the methods de-

scribed in the Stockholm papyrus. The im-

portance of calcium salts in dyeing with madder

was already recognized by Hausmann in 1791

(21), but the chemical mechanism of this calcium

effect on the dyeing of alum-mordanted material

with madder or alizarin was finally clarified only

in the 20th century.

Early chemical studies of alizarin-calcium

compounds: The history of alizarin was re-

viewed by Graebe and Liebermann (20). Alizarin

and purpurin were first isolated from madder

roots by Colin and Robiquet in 1826. Incidentally,

the name alizarin was derived from alizari, the

term for roots of Rubia tinctorum imported from

the Orient. The composition of alizarin was

determined independently by Strecker and by

Graebe and Liebermann in 1868 (20). The

calcium, barium and lead salts of alizarin were

clearly described by Graebe and Liebermann (20).

Perkin (35) published a detailed study of the Na,

Na,, K, K, and Ca salts of alizarin and of the

role of the 1- and 2-OH groups in salt formation;

he was apparently the first to suggest that in

alizarin-aluminum-calcium compounds calcium

“neutralized” the 2-OH group. These early

authors distinguished clearly between calcium

salts of alizarin and the lakes (chelates) of this

dye with aluminum.

The interactions of alizarin and other anthra-

quinones with metals and bases were studied in

detail by Pfeiffer (36, 37). Weak bases formed

TABLE II

Dissociation Constants of 1- and i-OH

Groups of Anthraquinones (p4)

2-Hydroxyanthraquinone 24 X 10-’Alizarin, first constant 6.6 X 10’

1-Hydroxyanthraquinone 3.2 X 10�’

Alizarin, second constant 1.1 X 10-12

salts only with the 2-OH group; alkali and earth

alkali hydroxides reacted first with the 2-OH and

then with the 1-OH group of ali.zarin. Only the

1-OH group participated in complex (chelate)

formation; the 2-OH group remained free and

could form salts. On the basis of these observa-

tions Pfeiffer (37) suggested that in dyeing with

madder or alizarin aluminum, iron, chromium

and similar metals form a complex with the

1-OH and 9-CO group; calcium and other earth

alkalis form a salt with the 2-OH group. The

physical chemistry of alizarin and other anthra-

quinone derivatives was studied by Huttig (24).

Comparison of the dissociation constants of

alizarin with those of 1- and 2-hydroxyanthra-

quinone indicated that the 2-OH group is more

strongly acid than the 1-OH group (Table II), as

already suggested by Pfeiffer (37). Correlating the

physical-chemical data with the experiences of

textile dyers, Huttig (24) suggested that salt

formation of a free hydroxy group with calcium

plays a major role in the formation of the

calcium-aluminum-alizarin compound. This con-

clusion was confirmed by Attree and Perkin (2),

Venkataraman (48), Harms (22), Gerstner (16)

and Kiel and Heertjes (25, 26).

Early histologic uses of madder and

alizarin: The literature from 1567 to the early

20th century on coloration of bones and/or

teeth of animals fed madder has been reviewed

by Cameron (8). The recognition of the relation

between coloration of bone by madder and

staining of calcium has been ascribed to Gottlieb

in 1914 (8). However, according to Haller (21),

Bancroft noticed this relation in 1818 and tried

to use calcium salts-without alum-as a

mordant in madder dyeing. The affinity of

madder for calcium salts of bones was known also

to 19th century histologists. In a discussion of

Lieberkuhn’s paper (1874, original not available),

Gierke (17) stated explicitly that after pigeons

had been fed madder the dye combined with the

calcium salts of bones, but not with the organic

matrix; the latter could be removed by boiling




00/\

00

0


Fzo. 1. Formula of the calcium-aluminurn-alizarin compound suggested by Rutishauser (22,45).

in NaOH without loss of coloration (“Nach

Futterung von Tauben mit Krapp verband sich

der Farbstoff mit den Kalksalzen der Knochen

und nicht mit der organischen Grundsubstanz

derselben. Man kann diese daher durch Kochen

in Natronlosung entfernen, ohne dass die Farbung

leidet.”).

Alizarin was apparently first used as a histo-

chemical reagent by LieberkUhn in 1874; he

injected a 5% neutral solution of alizarin and

reported reaction of the dye with calcium phos-

phate but not with calcium carbonate (17).

Despite these observations, Gierke (17) recom-

mended alizarin only for staining of spinal cord.

In the chapter on calcium salts in the Enzyklopa-

die der mikroskopischen Technik, Magnus (30)

discussed the role of calcium salts in textile

dyeing with alizarin and stated explicity that

calcium forms a salt with alizarin, yet, in the

chapter on alizarin, Magnus (30) remarked that

this dye was of little use in histologic technique;

alizarin red S was mentioned as a stain for

neuroglia, mitochondria and spinal cord. How-

ever, Roehl (43) obtained intense violet coloration

of calcium deposits in kidneys with alizarin in a

solution of NaOH; for staining with alizarin red S

he recommended addition of NaOH, ammonia or

lithium carbonate to the dye solution. These

early authors apparently regarded the calcium-

alizarin compound as a salt. The term lake for

the alizarin-calcium compound in tissue sections

seems to have been derived from Pfeiffer’s (37)

term “mixed lakes” (gemischte Lacke) for calcium-

aluminum-alizarin and similar compounds (5).

However, Pfeiffer’s (37) sharp distinction be-

tween calcium salt and aluminum, iron or

chromium chelate in these compounds became

blurred in the histologic literature, as shown by

Becher’s (5) complaints that in histology the

terms mordant and lake were not based on a

rational definition and were used much more

loosely and vaguely than in the textile industry.

Becher (5) tried to introduce the distinction

between alizarin-calcium salts and chelates with

aluminum and other metals into histology, but

apparently with little success. In the more

recent histologic literature available to us, this

difference is stressed only by Harms (22).

Recent chemical and infrared spectro-

scopic studies of alizarin: A rather complicated

structural formula of the calcium-aluminum-

alizarin compound (Fig. 1) was suggested

by Rutishauser in 1940 (22, 45). Parenthet-

ically, a similar structure has been suggested

for carmin, a calcium-aluminum compound of

carminic acid (22). However, in this structure

(Fig. 1) there would be a large strain in the

central bridge between the two aluminum atoms

(25). Kiel and Heertjes (25-28) therefore re-

investigated the composition and structure of

compounds formed by alizarin and its 3-deriva-

tives with calcium, aluminum and various other

0

�i�Lo-c0-�o

o�. ,o ___

H2OmAI 0-Ca---’O A

H20

Ca-0’

0

1”H20




Ca++‘‘H20

H20

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

TABLE III

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


H0-AI#{149}” H20,,�%

�IIIIIIX:r

Compound

Absorption Frequencies

2-OH 1-OH 10-CO 9-CO Complex Mo�tic �

AlizarinK-alizarin

Ca-alizarinCa-Al-alizarin

3-NO,-alizarinCa-3-NO,-alizarinCa-Al-3-NO,-alizarin

Alizarin red SCa-Al-alizarin red S

3400

3440

-�

3100-2800

3450

3050

-#{176}

cm’

1659 1630

1623

16121640

1678 16471649

16521700 16701650

1525

1505

1534

1583, 14561581, 14621575, 14701588, 1460

1590, 1452

15901578, 14721618, 1462

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-

aluminum alizarin compound contained one atom

of calcium, one atom of aluminum and two

alizarin residues (Fig. 2) (25). In other series

aluminum was replaced by iron or chromium;

sodium, potassium, barium or tin was substituted

for calcium. These compounds showed the same

proportions as calcium-aluminum-alizarin (26).

Electrolytic experiments demonstrated that

calcium isbound in the molecule by an ionic bond,

whereas aluminum, at least partly, is bound by a

covalent bond; i.e., it forms part of a chelate

ring. These conclusions were supported by

infrared spectroscopic studies (Table Ill) (27,

28). Furthermore, in the NaCl region the spectra

of potassium-aluminum-alizarin, barium-alumi-

num-alizarin, Sn(IV) -aluminum-ali.zarin, calcium-

chromium-alizarin and calcium-iron-alizarin were

identical with that of calcium-alurninum-alizarin,

indicating that these compounds all have es-

sentially the same structure (Fig. 2); i.e., they

are ionized salts of aluminum-, iron- or chromium-

alizarate (26).

The reaction of alizarin with the chelate-form-




Ca� +

0 OH

0

+H20


Fio. 3. Formation of the calcium salt of alizarin suggested by Kiel and Heertjes (28) on thebasis of spectroscopic data.

TABLE IV

Maxima of Absorption Bands in Ultraviolet

and Visible Spectrum (p8)

Compound Absorption bands

‘nih

AlizarinK-alizarin

332

350

(she) 434

570K,-alizarin

Ca-alizarin

Ca-Al-alizarin

332365

329

(sh) 593580

512

637625 (sh)

3-N02-alizarin 328 425K-3-NO2-alizarin

K,-3-NO2-alizarin

Ca-3-NO2-alizarin

351

361

364

(sh)(sh)

458529

520

568

562

(sh)

(sh)

a Shoulder.

ing metal aluminum alone was exceedingly slow.

Furthermore, the intensity of the 0-Al absorption

band was much lower than that of the 0-Al band

in the other compounds studied. The extremely

slow reaction of alizarin with aluminum was

ascribed to the strong hydrogen bond between

the 1-OH group and the 9-CO group; for a

complex to be formed, this hydrogen bond has to

be broken or at least weakened. This occurs when

salt formation at the 2-OH group takes place

before chelate formation (26, 28). These chemical

and spectroscopic data also explain the ex-

periences of textile dyers that hard water or

addition of chalk is essential in alizarin (Turkey

Red) dyeing. According to Becher (5) and Harms

(22), in the absence of calcium alizarin stains

tissues mordanted with aluminum yellowish, and

the stain is not fast (ganz unecht); obviously,

chelate formation does not take place under this

condition.

Kid and Heertjes (28) also studied the

structure of potassium and calcium salts of

alizarin in the absence of chelate-forming metals.

The infrared spectrum of the monobasic salt

potassium 2-alizarate had an absorption band at

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

this band could not be due to water, it had to be

ascribed to the 1-OH group. Kiel and Heertjes

(28) therefore concluded that in potassium 2-

alizarate the 1-OH group no longer forms a

hydrogen bridge with the 9-CO group, or the

bridge has been considerably weakened. This must

be caused by salt formation at the 2-OH group.

Since the 2-OK� group is in conjugation with

with the 10-CO group, the frequency of the

latter group must be lowered, and the band at

1551 cm’ has therefore been ascribed to stretch-

ing of the 10-CO group. The bathochromic shift

of the absorption bands of alizarin in the visible

spectrum after salt formation is also ascribed to

this resonance (Table IV) (28).

The formation of the calcium salt of alizarin

is shown in figure 3 (28). The proposed structure

of calcium-alizarate is supported by the following

spectroscopic data. In the visible region the

spectra of calcium-alizarate and K2-alizarate

are similar (Table IV); since the two K� ions

can only be bound to the 1- and 2-0- groups,

the bivalent Ca� should be similarly bound.

Because calcium-alizarate shows only one

stretching frequency at 1612 cm’, both -0-

Ca� bonds must be equivalent. Because no

bands could be found at about 1525 cnc’ (com-

plex ring vibration) and at about 3500 cm� (OH

stretching frequency), formation of a calcium-

alizarin complex (chelate) could be ruled out (28).

These spectroscopic observations confirm earlier

chemical findings discussed above, which indi-

cated that the alizarin-calcium compound is a

salt (2, 19, 35-37).

It may be objected that the marked color

change of alizarin upon interaction with calcium

or various other cations indicates complex forma-

tion rather than salt type linkages. Valko (47)

ascribed the color change upon salt formation to

the polarizing effect of the calcium ion. As

already mentioned, Kiel and Heertjes (27)





demonstrated that such bathochronic shifts are

due to alterations of the resonance system of

alizarin during salt formation (28) . Moreover,

alizarin and alizarin red S are polygenetic dyes;

i.e., the color of the dye salt varies with the

cation (31, 38, 40, 48). Color differences among

various metal compounds of alizarin and alizarin

red S were observed also in the model experi-

ments described above (Table I), but these color

changes do not permit conclusions whether

certain metals are bound by salt type linkage or

by chelate formation.

Correlation of chemical data and staining

properties of alizarin: As shown in Table II,

the dissociation constant of the 2-OH group of

alizarin is 6.6 x 10-’ and that of the 1-OH group

is 1.1 x 10” (24); the corresponding pK values

are 8.18 and 11.96 respectively. Therefore,

alizarin in aqueous solutions of pH 7 or below is

only slightly dissociated and shows little tend-

ency to form calcium salts. This also explains

the faint coloration of bones in in vivo experiments

observed by Richter (42). Differences between in

vivo staining with alizarin and madder are

discussed below. In solutions of alizarin adjusted

to pH 9 about 50% of the 2-OH groups should be

dissociated; the red coloration of calcium de-

posits by such solutions indicates formation of themonoalizarate. Around pH 12 nearly all 2-OH

and about half of the 1-OH groups are dissociated

and the blue dializarate is formed. The reddish

blue coloration of calcium deposits suggests

that the red monoalizarate and the blue di-

alizarate may occur side by side. That the

calcium-alizarin compound in tissue sections is a

salt, and not a chelate, is indicated by its sensi-

tivity toward dilute acetic acid. As pointed out

by Becher (5), Cameron (8) and Harms (22),

calcium salts of alizarin dissociate readily in cold

acetic acid, whereas alizarin-metal chelates

disintegrate only after prolonged boiling in

H2SO4 or other mineral acids.

Unfortunately, alizarin is not specific for

calcium but can react also with other metals

(Table I). In “blind” tests the color differences

among various alizarin-metal compounds were

found insufficient for identification of the cations.

Identification is complicated also by the fact

that the anion of the salt can affect the color of

the alizarin-metal compound as shown by the

calcium and iron salts in Table I. This effect

seems to be due, at least in part, to alterations of

the pH of the dye solution by the anion. For

example, Ca(H2PO4)2 did not form colored

compounds with alizarin in pH 9 buffer solutions

or in 1 % NaOH (Table I). As already mentioned,

saturated solutions of Ca(H2PO4)2 in these

solvents had pH values of 2-2.5. Obviously, the

hydroxyl groups of alizarin are not dissociated

under these conditions and salt formation cannot

take place. If a calcium deposit should consist

entirely of Ca(H2PO4),, it may remain unstained.

Hence, alizarin cannot be depended on to visualize

primary calcium phosphate.

Madder versus alizarin: Differences between

madder and synthetic alizarin in vital staining

of bone have been stressed by Richter (42).

According to the Colour Index (9), madder

contains six dyes in the form of their glycosides,

namely alizarin (rubierythric acid, CI. 75330),

xanthopurpurin (1 ,3-dthydroxyanthraquinone,

C.I. 75340), xanthopurpurin-3-carboxylic acid

(C.I. 75370), rubiadin (1 ,3-hydroxy-3-methyl-

anthraquinone, C.I. 75350), purpurin (1,2,4-

hydroxyanthraquinone, C.I. 75410) and pseudo-

purpurin (purpurin-3-carboxylic acid, C.I. 75420).

Comparative in vivo studies with alizarin and

purpurin-3-carboxylic acid showed that only the

latter gave the bright carmine red color typical

of madder-stained bones; alizarin tinted bones

slightly pale bluish pink; extraction of madder-

stained bones confirmed that coloring of bone

was mainly due to purpurin-3-carboxylic acid

(42). In in vitro experiments, purpurin-3-carbox-

ylic acid formed colored calcium salts in the acid,

neutral and alkaline range (42). Unfortunately,

xanthopurpurmn-3-carboxylic acid and purpurmn-3..

carboxylic acid were not available for this study.

However, the data provided by Richter (42)

indicate that the staining properties of purpurin-

3-carboxylic acid are similar to those of alizarin-3-

sulfonic acid. Thus, staining patterns obtained

with madder should be compared with those

produced by alizarin red S rather than by

alizarin.

Interaction of alizarin red S and other

sulfonated dyes with calcium: The 1- and

2-OH groups of alizarin red S show the same

behavior toward calcium and various other

metals as those of the parent dye alizarin (27, 28).

Differences between frequencies of certain bands

in the visible and infrared spectrum of salts of

alizarin and its 3-substituted derivative have

been ascribed to effects of the electronegative

nitro or sulfonic acid group (27). However, the

interaction of alizarin red S with calcium is not





limited to the 1- and 2-OH groups; the sulfonic

acid group also forms a salt with calcium. One

calcium atom can react with two sulfonic acid

groups; i.e., it can bind two dye molecules of

alizarin red S according to the general formula:

2 dye-SO3Na + CaC12 -� (dye-SO,)2Ca + 2

NaCl (4, 40). Salt formation between alizarin

red S and calcium, strantium, barium, zinc,

magnesium, aluminum and other metals later

than group II in acid solutions was described

by Atack (1), who emphasized that these com-

pounds are salts, not complexes.

Formation of insoluble or poorly soluble

calcium or barium salts of sulfonated dyes is

widely used in industry in the preparation of

pigments; these dye salts are referred to as lakes

or toners. In the dye and pigment industry

the term lake denotes a poorly soluble or insolu-

ble salt of a water-soluble acid or basic dye (31,

38, 41, 48). Sulfonated dyes suitable for this

purpose are classified as “acid dyes for lakes”

(10). In the precise terminology of dye chemistry

the designation “acid dye” signifies that these

dyes do not contain chelating groups. Dyes

capable of chelate formation with metals are

classified as mordant dyes. Of course, certain

mordant dyes can also form lakes, i.e. the readily

soluble sodium or potassium salts can be con-

verted into poorly soluble calcium or barium

salts. Barium salts (lakes) of sulfonated anthra-

quinone dyes have been described by Mattiello

(31). Alizarin red S is now mainly used in the

preparation of lakes; in textile dyeing it has been

largely replaced by other dyes (4).

The effect of pH on lake formation by acid

dyes has been discussed by Pratt (38). In these

chemical studies an excess of metal was used.

At pH 5.5 and below, all dye molecules reacted

with the metal. Lake (salt) formation decreased

around pH �; in alkaline solutions only a mod-

erate amount of dye was bound by the metal.

This pH effect is very inconvenient for histology.

At pH 5.5 and below, basic groups of tissues also

react with sulfonic acid groups of dyes; this

diffuse coloration of sections made identification

of small calcium deposits very difficult. The

choice of acid dyes for lakes is therefore limited

to those dyes which produce strong selective

coloration of calcium deposits in the nonoptimal

pH range 7-9.

In contrast to acid dyes for lakes, hydroxy-

anthraquinone dyes containing acid groups can

readily form salts over a wide p11 range. In

acid and neutral media only the sulfonic or

carboxylic groups take part in salt formation

(42). Acid and neutral solutions of alizarin red

S are ochre yellow; the salts (lakes) formed with

calcium in model experiments and in calcifiedtissues are colored orange or yellowish red.

In alkaline solutions the hydroxyl groups partici-

pate in salt formation (42); the calcium salts

formed under these conditions are deep red to

bluish red. The bluish tinge becomes more

noticeable with increasing PH; i.e., the salts of

alizarin red S formed in strongly alkaline solu-

tions resemble those of alizarin. In the weakly

alkaline region (around pH 9) the two types of

reactions probably overlap. This assumption

is supported by observations that at pH 9

calcium deposits are stained selectively by acid

dyes for lakes and by alizarin and alizarin blue

S. In analogy to the formula for the calcium-

aluminum compound of alizarin red S given by

Baumann and Hensel (4), the calcium salt

formed under alkaline conditions would be (Ca-

ali.zarin-3-SO3)2Ca. As in the case of alizarin,

the color change of alizarin red S cannot be

regarded as evidence for complex (chelate)

formation. Striking color changes upon salt

formation with calcium or barium have also

been observed with acid dyes for lakes which

do not contain chelating groups (31).

The use of the term lake as a synonym for

chelates apparently caused some ambiguity

even in the chemical literature. For example,

compounds of alizarin red S with hafnium and

other cations of the titan group have been in-

terpreted as chelates (innere Kompl&xsalze) (12),

yet Venkataraman (48) cited spectroscopic

evidence that the hafniurn-alizarin compound

is a salt, not a chelate. Hence, reports concerning

complex (chelate) formation of alizarin and its

derivatives with various cations should be

interpreted with caution. The investigations

by Kiel and Heertjes (25-28) indicate that

infrared spectroscopy is at present the most

reliable method for distinction between salts

and chelates of alizarin and its derivatives.

Alizarin red S is as nonspecific for calcium as

the parent dye alizarin. These observations artin agreement with findings by Dahl (11). Im

addition to the compounds listed in Table I�

alizarin red S is also very sensitive towart

the ions of uranium, titanium, bismuth ank

thallium (15), and toward zirconium, hafnium

and thorium (12). The differences in color





among various alizarin red S-metal compounds

tested were insufficient for identification of

the cations in a blind study. As already men-

tioned, the same difficulty was encountered with

alizarin.Binding of acid dyes for lakes by calcium

deposits in tissues: The selective coloration

of calcium deposits by several acid dyes for

lakes shows that salt formation between sul-

fonic acid groups and calcium or other cations

occurs under the conditions of histologic tech-

nique. The abolition of staining by pretreatment

of sections with dilute acetic acid substantiates

the conclusion that these dyes react with cal-

cium or other metallic cations and not with

basic groups of proteins. Preliminary compara-

tive studies of acid dyes for lakes and other

acid dyes indicate relations between the configura-

tion of dye molecules and selectivity for calcium

deposits. Even dyes which differ only in the

position of one sulfonic acid group can show

strikingly different staining properties. For

example, the monoazo dye Bordeaux red B

(1-naphthylamine -‘ 2-naphthol-3 , 6-disulfonic

acid) colored calcium deposits strongly and

selectively, but Ponceau 6R (1-naphthylamine

2-naphthol-6 ,8-disulfonic acid) showed no affinity

for calcium deposits. Further investigations of

the relations between dye structure and affinity

for calcium are in progress. Previous studies

showed relations between fluorescence and

structure of azo dyes (39). These relations were

found to apply also to the calcium lakes. Of the

dyes tested, Ponceau 2R was found most suitable

for fluorescence microscopic studies of calcium

deposits.

The effects of different fixatives and of varia-

tions in fixation and embedding procedures on the

binding of acid dyes for lakes and the specificity

of these dyes for various cations have not yet

been determined. These dyes were included in this

study for the sole purpose of obtaining informa-

tion concerning the role of the sulfonic acid group

of alizarin red S in the staining of calcium deposits

in alcohol- or Carnoy-fixed tissues. Therefore,

until further data become available, these dyes

should not be regarded as substitutes for alizarin

and its derivatives.

Binding of alizarin red S by tissue sections:

The recommended pH values for solutions of

alizarin red S vary from 4.1-4.2 (32) to 6.3-6.5

(11). Our experiences indicate that the staining

patterns obtained with alizarin red S in acid

solutions differ significantly from batch to batch

and apparently depend on the impurities in the

dye samples. Of the five samples tested in this

study, only two produced passable selective

staining of calcium deposits at pH values below

7. The yellow impurities of the other three dye

samples competed effectively with alizarin red

S for binding sites in calcium deposits. Thus the

discrepancies between recommended pH values

and reported difficulties in reproducing the work

of others can readily be explained by differences

in the dye samples used by various workers. The

low pH values of aqueous solutions of the impure

dyes and the staining properties of the con-

taminants indicate that these compounds are

strongly acid. Since many commercial samples of

azo dyes contain intermediates (44), it seemedconceivable that batches of alizarin red S may

also be contaminated with sulfonated by-

products of the manufacturing process. According

to Fierz-David and Blangey (13) and Venkatara-

man (48), 2-anthraquinonesulfonic acid is used as

starting material in commercial production of

alizarin. A concentrated solution of 2-anthra-

quinonesulfonic acid, NaOH and NaC1O3 or

NaNO3 is heated at 185#{176}Cand 5-6 atm pressurefor 48-72 hr. The 2-anthraquinonesulfonic acid

is converted into 1-hydroxyantb.raquinone-2-

sulfonic acid; in a second step the disodium salt of

alizarin is formed. Completion of the reaction is

indicated by loss of fluorescence; 2-anthra-

quinonesulfonic acid and 1-hydroxyanthra-

quinone-2-sulfonic acid are strongly fluorescent,

but alizarin is nonfluorescent (13). Alizarin red S

is prepared by sulfonation of alizarin. When

tissue sections stained with impure samples of

alizarin red S were viewed in ultraviolet blue

light, the structures colored by the yellow

contaminants showed moderate to strong yellow-

ish red fluorescence. Calcium deposits stained red

by alizarin red S were nonfluorescent. Theseobservations suggest that the yellow impurities

consist, at least in part, of intermediates.

Further disadvantages of acid solutions of

alizarin red S are diffusion artifacts and loss of

calcium. Gomori (19) recommended buffer solu-

tions of pH 4.5-5 for removal of calcifications in

tissues. It is therefore not surprising that solutions

of alizarin red S at pH 4.8 or below removed

significant amounts of calcium, and diffusion

artifacts became evident within 1 mm. Hence,

acid solutions of alizarin red S are unsuitable for

studies of the localization of calcium deposits at





the intracellular level, e.g., in muscle fibers or

renal epithelial cells, and for semiquantitative

estimates. In the discussion of methods for

enzymes, Gomori (19) stated explicitly that

calcium salts cannot be used effectively below pH

8.5 because of their solubility and consequent

diffusion artifacts. Such diffusion artifacts were

very obvious in sections stained with alizarin red

S at pH 7.2, but were absent in sections stained

with alizarin red S at pH 9, the pH value recom-

mended by Gomori (19) for procedures employing

calcium salts. Parenthetically, alkaline solutions

of alizarin red S were already in use around the

turn of the century. Roehl (43) recommended

differentiation in alkaline media or addition of

NaOH, Li2CO3 or NH3 to aqueous solutions of

alizarin red S. Differentiation is unacceptable in

histochemistry (34) and impractical for general

hospital pathology. In this study, 0.5% solutions

of alizarin red S buffered to approximately pH 9

yielded optimal coloration of calcium deposits. As

already mentioned, at this pH the yellow im-

purities present in three dye batches were no

longer bound by tissue sections; the staining

patterns obtained with the five dye batches

tested were indistinguishable. Higher p1-1 values

were found impractical because the sections

tended to become detached from the slides.

Because solutions of alizarin red S buffered to

pH 9 do not cause noticeable diffusion artifacts

or loss of calcium salts, staining can be prolonged

to 1 hr. Intensity of coloration of calcium,

aluminum or other salts by alizarin red S in-

creases with the time allowed for lake (salt)

formation (49). Since replacement of anions in

calcium deposits by the dye progresses gradually

from the periphery, staining for 30-60 mm

improved coloration of crystalline areas more

than 10 � in diameter. The slight grayish pink

background stain did not interfere with the study

of small intracellular calcium deposits and

actually served as a counterstain. As pointed out

by Barka and Anderson (3), counterstaining is

undesirable from a histochemical standpoint.

Because calcium and other salts of alizarin red S

are soluble in slightly acid media (33), acid dyes

are difinitely unsuitable as counterstains. Basic

dyes tend to react with calcium deposits and are

therefore not recommended (29).

Effect of fixatives: The solubility of calcium

deposits in solutions of pH 4.5-5 (19) readily

explains the deleterious effect of unbuffered 10%

formalin. Treatment of alcohol- or Carnov-fixed

sections with 10% unbuffered formaliti for 15 hr

removed all calcium deposits. Similar treatment

�vith Zenker-formol (Spuler, �Iaxiiuow) was

equally effective. Ilence, the major loss of

calcium seems to occur (luring fixation and not

at later stages of the embeddmg procedure.

According to 1)ahl (11), solutions of formaliti

buffered to I)11 7.2 or 8.35-8.7 also caused

significant loss of calcium salts and were clearly

inferior to al)solute alcohol. Obviously, these

fixatives are unsuitable for histochemical studies

of calcium deposits. That (‘arnoy’s fluid, which

contaim 10% acetic acid, preserves calciuni

deposits as wellas absolute alcohol is probably due

to the very low dissociation of acetic acid and pOOr

solubility of calcium salts in absolute alcohol and

chloroform.

REFEHENCES

I. Atack, F. W.: A new reagent for the detectionand colorimetric estimation of aluminum.

J. Soc. Chem. md. 34: 936, 1915.2. Attree, G. F. and Perkin, A. G.: Reduction

products of the hydroxyanthraquinones.J. Chem. Soc. 134: 144, 1931.

3. Barka, T. and Anderson, P. J.: Histochemistry:Theory, Practice and Bibliography. HoeberMedical Division, Harper and Row, Pub-ushers, Inc., New York, 1963.

4. Baumann, 11. and ilensel, II. H.: Neue Metal!-komplexfarbstoffe, Struktur und farberischeEigenschaften. For(seh r. Chc?n. Forsch. 7:

643, 1967.5. Becher, S.: Untersuchungen liber Echtfarbung

der Zeilkerne mit k#{252}n.stlichen Beizenfarb-stoffen und die Theorie des hislologischenFarbeprozesses mit gelosten Lacken. Verlagvon Gebruder Borntraeger, Berlin, 1921.

6. Caley, E. II.: The Leyden Papyrus X. AnEnglish translatioii with brief notes. .1.Chem. Ed. 3: 1149, 1926.

7. Caley, E. R.: The Stockholm Papyrus. AnEnglish translation with brief notes. .1.Chem. Ed. 4: 979, 1927.

8. Cameron, U. H.: The staining of calcium.J. Path. Bact. 33: 931, 1930.

9. Colour In(iex, Ed. 2, Vol. 1. Society of 1)yers

and Colourists and American Associationof Textile Chemists and Colorists, Lowell,Mass., 1956.

10. Colour Index, Ed. 2, Vol. 2. Society of 1)yersand Colourists and American Associationof Textile Chemists and Colorists, Lowell,Mass., 1957.

11. 1)ahl, L. K.: A simple and sensitive histo-chemical method for calcium. Proc. Soc.Exp. Biol. 80: 474, 1952.

12. l)orta-Schaeppi, Y., ilurzeler, H. arid Tread-well, W. 1).: Uber die St#{246}chiometrie undExtinktion geloster Alizarinlacke von Ka-tionen der Titanreihe. Helv. Chim. �tcta34: 797, 1951.

13. Fierz-David, H. E. and Blangey, L.: Grund-legende Operationen der Farbenehemie, Ed.8. Springer \Tcrlag, Wien, 1952.




124 PUCHTLEII, MELOAN AND TERRY

14. F’orhes, H. J. : Chemical, culinary and cosmeticarts. In .1 History of Technology, editedby C. Singer, E. J. Holmyard and A. ii.hail, vol. 1. Oxford University Press, New\ork, 1954, i. 238.

15. (;erii�uth, F. (1. and �1itchell, C. : I)etectioii

itII(l identification of specific cations withso(liun�-alizarili-sulfonate. .1 iiier. J. Pharm.101: 46, 1929.

16. Gerstner, ii. : Die (‘heinie (icr .1pplikation vonKorn plcxfarbstoffen. Akademie Verlag, Ber-liii, 1959.

17. Gierke, 11.: Fiirberei zu mikroskopischenZwecken. Z. Wiss. Miki. 1: 62, 497, 1884.

18. Cierke, II.: F#{228}rberei zu mikroskopischeii

Zwecken. Z. Wis.s. Mikr. 2: 13, 1885.19. Gomori, (4.: Microscopic Histochemistry.

University of Chicago Press, Chicago, 1952.20. (lraebe, C. and Liebermaiiii, C.: Ueber

Anthracen mid Alizarin. A nn. Chem.Pharm., �‘�P1�1. 7, 257, 1870.

21. llaller, H.: Vorn T#{252}rkischrot zum Alizarinrot.Melliand Textilhei. 19: 448, 504, 595, 731,796, 1938.

22. harms, II.: Handbuch dci Farbstoffe liii dieMikroskopie, Teil II, 1. Liefg. StaufenVerlag, Kamp-Lintfort, 1957.

23. ll#{252}bner, J.: A contribution to the history of

dyeing. J. Soc. Dyers Go!. 29: 344, 1913.24. llQttig, U. F.: Physikalisch-chemische Unter-

suchungen (ler ()xy - mid 1) ioxyanthra-chinone ni it besonderer Berucksichtigungihres Beizvermbgens. Z. Phy.s. C/rein. 87:129, 1914.

25. Kiel, E. U. and Heertjes, P. M.: Metal com-plexes of alizarin. I. The structure of thecalcium-aluminum lake of alizarin. J. Soc.Dyers Go!. 79: 21, 1963.

26. KieI, E. U. and lleertjes, P. M.: Metal corn-plexes of alizarin. II. The structure of somemetal complexes of alizarin other thanTurkey red. J. Soc. Dyers Go!. 79: 61, 1963.

27. Kid, E. U. and lleertjes, P. M.: Metal corn-plexes of alizarin. III. The structure ofmetal complexes of sonic 3-derivatives of

alizarin. J. Soc. Dyers Go!. 79: 186, 1963.28. Kiel, E. U. and Heertjes, P. M.: Metal com-

plexes of alizarin. IV. The structure of thepotassium and calcium salts of alizarinand 3-nitroalizariti. J. Soc. I)�jeis Col. 79:363, 1963.

29. Lillie, R. I).: Histopathologic Technic andPractical Histocheinistry, Ed. 3. The Blakis-ton 1)ivision, McUraw-H ill Book Company,New York, 1965.

30. Magiius, \V.: Alizariti, Alizaritie, Calciumace-tat. In Enzyklopadie (icr MikroskopischenTechnik, edited by P. Ehrlich, H. Krause.M. Mosse, H. Hosin and K. Weigert, Ed. 2,Vol. 1. Urban and Schwarzeiiherg, Berlin,1910, pp. 14 and 161.

:31. Mattiello, J. J.: Cheiiiistrv of tie synthetic

organic �)igmeIlts. In Protective and Decora-live Goatings, edited by J. J. Mattiello, Vol.2. John Wiley and Sons, New York, 1942.

32. McGee-Russell, S. M. : Histochemical methodsfor calcium. J. Histochem. Cytochem. 6: 22,1958.

33. Parker, C. A. and Goddard, A. P. : The reac-tion of aluminum ions with alizarin-3-sulfonate with particular reference to theeffect of addition of calcium ions. Anal.Chin,. Acta 4: 517, 1950.

34. Pearse, A. G. E.: Histochemistry: Theoreticaland Applied, Ed. 2. Little, Brown andCompany, Boston, 1961.

35. Perkin, A. U.: A reaction of some phenoliccolouring matters. J. C/rein. Soc. 75: 433,1899.

36. Pfeiffer, P.: Zur Kenntnis der Farblacke. I.Ber. I)tsch. C/rem. Ges. 44: 2653, 1911.

37. Pfeiffer, P.: Zur Theorie der Farblacke. II.Ann. Chem. 398: 137, 1913.

38. Pratt, L. S.: The Chemistry and Physics of

Organic Pigments. John Wiley and Sons,New York, 1947.

:39. Puchtler, H., Sweat, F. and Uropp, S.: Aninvestigation into the relation betweenstructure and fluorescence of azo dyes. J.Roy. .1! icr. Soc. 87: 309, 1967.

40. Hath, 11.: Lehrbuch der Textiichemie, Ed. 2.Springer-Verlag, Berlin, 1963.

41. Remington, J. S. and Francis, W.: Pigments:Their Manufacture, Properties and Use.Leonard Hill, Ltd., London, 1954.

42. Richter, 1).: Vital staining of bones withmadder. Biochem. J. 31: 591, 1937.

43. Hoehl, W.: Uber Kalkablagerung und-Ausscheidung in der Niere. Beitr. Path.Anat., suppl. 7, 456, 1905.

44. Rosenthal, S. I., Puchtler, H. and Sweat, F.:Paper chromatography of dyes: method toinvestigate vagaries in histological staining.Arch. Path. (Chicago) 80: 190, 1965.

45. Schweizer, H. H.: Kiinstliche organischeFarbstoffe und ihre Zwischenprodukte.Springer-Verlag, Berlin, 1964.

46. Taylor, F. S. and Singer, C.: Pre-scientificindustrial chemistry. In A History of Tech-nology, edited by C. Singer, E. J. Holmyard,A. R. Hall and T. I. Williams, Vol. 2. OxfordUniversity Press, New York, 1956.

47. Valk#{243},E.: Kolloidchen,ische Grundlagen derTextilveredlung. Springer-Verlag, Berlin,1937.

48. Venkatararnan, K.: The Chemistry of Syn-thetic Dyes, Vol. 2. Academic Press, NewYork, 1952.

49. \oe, J. H. and Hill, W. L.: An investigationof the reaction of sodiurn alizarin mono-sulfoiiate with aluminum under differentexperimental conditions with reference toits use in colorimetrv. J. Amer. Chem. Soc.50: 748, 1928.




Documents

On the History and Mechanism of Alizarin and Alizarin Red s Stains