Resumen por el autor, Oscar V. Batson.

La coloraci6n diferencial del hueso.

I. El teiiido de ejemplares conservados.

El autor ha probado una serie de derivados de la alizarina y

mordientes con el prop6sito de hallar, si esto fuese posible, un
colorante que ofrezca un8 coloraci6n de contraste con la obtenida
por el rojo de rubia ordinario. Para 10s experimentos ha em-
pleado embriones de pol10 y cerdo. El material fu6 fijado en
formol a1 10 por ciento. Para eliminar el color de la sangre se
emple6 una solucibn dbbil de per6xido de hidr6geno. La solu-
ci6n tint6rea fu6 una soluci6n saturada del colorante en alcohol
de 95", una parte, y veinte partes de alcohol de 95'. Los ejem-
plares fueron diferenciados en una soluci6n al medio por ciento
de Acid0 sulfdrico en alcohol de 95", despu6s de lo cual fueron
deshidratados y aclarados s e g h el m6todo de Spalteholz. Los
veinte colorantes que pudieron obtenerse fueron 10s siguientes:
Negro de alizarina Bcido, R. Pardo de alizarina RB, polvo,
Meister, Lucius & Briining Fr. Bayer & Co.
Negro de alizarina SR, polvo, Negro de alizarol 3G, Nat.
Fr. Bayer & Co. Aniline and Chemical Co.
Azul de alizarina BrSg, polvo, Carmin de indigo, Griibler
'

Fr. Bayer & Co.

Negro de alizarina Bayer NR, Acid0 carminico, Grubler
Polvo, Fr. Bayer & Co.
Negro de alizarina SBB, polvo, Rojo de Burdeos, Grubler
Fr. Bayer & Co.
Pardo de alizarina AR, polvo, Naranja, Grubler
Fr. Bayer & Co.
Translation by J d F. Nonidea
Cornell Medical College, New York
A W H O R ’ B ABBTRACT OF T H I B PAPER IBBUED BY
THE BIBLIOQRAPHIC BERVICE. BEFTEMBER 26

THE DIFFERENTIAL STAINING OF BONE

I. THE STAINING O F PRESERVED SPECIMENS’

OSCAR V. BATSON
Anatomical Laboratory, University of Wisconsin, Madison

The study of ossification centers, of healing in fractures, and of

bone growth in general has been greatly facilitated by the use of
alizarin in its natural and synthetic forms. That madder root
when fed to animals colors the bone red has long been known.
It seems that Belchier (1736) rediscovered this fact and brought it
to popular attention. Almost 150 years earlier, however, Lae-
vinius Lemnius (1581) mentioned this property of madder root.*
Considering the numerous editions and translations of Lemnius’
“Hidden Miracles of Nature,” it seems remarkable that no use
was made of this observation.
Biologists and chemists have examined madder root to find the
active staining principles. The chief of these have been found to be
alizarin and purpurin, mainly the former. Since the pure syn-
thetic alizarin has been available it has been largely used in the
staining of preserved specimens, for the demonstration of the
skeleton. This staining technic coupled with clearing methods
has been presented in detail by Lundvall ( ’04,’05) and Spalte-
holz (’14). Alizarin in the form of its soluble salt, sodium
alizarin monosulphonate, has also been used to supplant madder
root in the vital staining of bone. Gottlieb (’14), in a very
thorough paper, describes the use of the above salt for vital
staining. Brooks (’17, ’20) more recently has reported a series
1A series of specimens stained with t h e described dyes was demonstrated at
the meetings of t h e American Association of Anatomists at The Wistar Institute,
Philadelphia, March, 1921. This demonstration included a set t h a t had been
prepared in August, 1920.
2 “. ...erythrodanum seu rubea, qua ossa pecudum sandicino rubentique
colore imbuit, . . .”
D e Miraculis Occultis Naturae, p. 390, lines 30 and 31.
159
160 OSCAR V. BATSON

of studies in which the same salt was used, and it is quite probable
that sodium alizarin monosulphonate is now used in most labo-
ratories for vital staining of bone.
Graebe and Lieberman first produced alizarin synthetically in
1868. Since that time many synthetic alizarin derivatives have
been produced by the dye chemists. A glance at the catalogs of
the dye firms reveals alizarin reds, browns, blacks, blues, greens,
and yellows. Apparently none of these have been used for the
successful’staining of bone. Ehrlich (’85) used alizarin blue S in
studying oxidation in tissues, but with this stain did not report
any staining of bone. Rawitz (’96) who used alizarin extensively
in microscopic technic did not mention bone staining. Spalteholz
(’14) uses small quantities of alizarin cyanatum in staining pre-
served specimens to improve the color of the alizarin red. Gott-
lieb (’14) unsuccessfully tried t o stain bone intra vitam with
alizarin blue and alizarin green (brand or formula not men-
tioned) .3 I n reporting Bardeen’s technic for the study of ossifica-
tion centers, Hill (’06) describes the former’s use of alum carmine,
a dye of a wholly different chemical series. Miller (’21) in a re-
cent paper refers to the same work.
In certain types of bone work a stain affording a contrast to the
red-brown of the usual alizarin would seem desirable. It might be
possible to alternate the red with a contrasting coloi in intra vitam
work, or it might be possible to bring out more detail after pres-
ervation by the use of a counterstain for that part of the bone not
colored during life. With the view of possibly finding such a
stain, the following study of alizarin derivatives and mordant dyes
was begun.
Among the readily soluble alizarin cloth dyes available those
were selected which gave promise of affording a contrast to the
alizarin red. The non-alizarin mordant dyes of the histological
laboratory were tried out, partially to see if the anthraquinone
grouping (present in the alizarins) is an essential element of a bone
stain and partially in the hope of finding a more brilliant bone
stain.
“ I c h mochte hier noch kurz erwahnen, dass ich mit einer Reihe verwandter
Farbstoffe (Alizarinblau und Alizaringriin) nach dieser Richtung Versuche
angestellt habe, samtlicki aber negativ ausgefallen sind” (S. 192).
 DIFFERENTIAL STAINING O F BONE 161

I n investigating the properties of the various stains the follow-

ing technic was used. The tissue consisted of portions of pig
embryos ranging in size from 3.5 cm. t o 8 cm. These embryos
were part of the laboratory stock obtained from the packing house
fixed in 10 per cent liquor formaldehyde. The blood color was
removed from part of the material by bleaching in a per cent
by volume solution of hydrogen peroxide (the ordinary peroxide
of commerce is 3 per cent by volume), followed by removing the
gas bubbles with the suction pump.
Stock solution OJ stain
Strain being investigated.. .................................... 250 mg.
Water.. ..................................................... 5 cc.
Shake well and add of 95 per cent alcohol. ...................... 95 cc.

I n no case did all of the stain go into solution.

1. Material washed in water.
2. Stained twenty-four to forty-eight hours in
Stock solution. ............................................... 1 part
95 per cent alcohol. ......................................... 20 parts

3 . Differentiated in 3 per cent solution of sulphuric acid in

95 per cent alcohol.
4. Dehydrated and cleared after the method of Spalteholz.
T o the present time twelve stains have been found to combine
differentially with the bony skeletal elements. h list of the stains,
with a brief comment on their behavior and the results obtained,
is given below.
Alizarin group
1. Acid alizarin black R. Farbwerke vorm. Meister, Lucius, &
Briining. Hoechst o/M. Stains the skeletal elements a deep reddish
puaple. The soft tissues and cartilages decolorize rather slowly;
however, differentiation is good from the beginning.
2. Alizarin-black SR powder. Farhen fabriken vorm. Fr. Bayer &
Co., Elberfeld. This stain likewise imparts a purple color to the boncs.
Satisfactory stains may be obtained from 1 to 250 dilutions. When
this is done thc bone is not stained so intcnsclp, hut decolorization is
easier. Must be differentiated in sulphuric acid alcohol.
3. Alizarin-blue Br3G powder. B a y r . Stain is fairly differential
from the start. Bone is an azure blue.
162 OSCAR V. BATSON

4. Alizarin-black-Bayer NR powder. Bayer. This stain gives a

good purple t o the bones. The other tissues decolorize readily and
the end-result is a very sharp stain. See no. 8.
5 . Alizarin black SBB powder. Bayer. Bones a sharp indigo.
The other tissues take this stain slightly. The differentiation is rapid
and sharp. This stain seems to be one of the most satisfactory. Its
color is devoid of reddish tinge.
6. Alizarin brown A R powder. Bayer. Imparts a reddish brown to
bone.
7. Alizarin brown R B powder. Bayer. Very similar to no. 6.
8. Alizarol black 3 G. National Analine and Chemical Co., New York.
A new American stain, supposedly an alizarin. The shade is slightly
different from no. 4, but otherwise the dye is quite similar. This is a
very satisfactory stain. It is available in quantities.
9. Indigocarmine, Grubler. This stain can be used in a saturated
solution or in any dilution. It decolorizes perfectly in water as well as
in the acid alcohol mentioned. The resulting stain is a medium indigo.
One of the easiest stains of the series to handle and one produeing an
excellent preparation.
10. Carminic acid, Grubler. This stain like the previous one can be
differentiated in water. If the reaction of the mounting fluid is acid the
color of the bone in the specimen is a n orange-red, if the reaction is
alkaline the color approaches a Bordeaux. If acid decolorixation is
used the specimen should be alkalinized before mounting. Dr. Bar-
deen’s use of alum carmine was referred to earlier. It is perfectly obvious
that the use of carminic acid is but a modification of this technic.
11. Bordeaux red, Grubler. This stain can be used in a saturated
solution if desired, but this makes decolorization a little more tedious.
The skeletal elements come out a brilliant shade of Bordeaux and if
decolorized properly the rest of the structures are colorless. The end-
results seems much better than those obtained with the usual alizarin
red.
12. Aurantia, Grubler. Solutions near saturation work best. The
bones stain a bright orange-yellow. The crystalline stain gives a deeper
stain than the amorphous.

I n working with the individual stains certain modifications as

noted above were found to be desirable, but on the whole the
standard technic was entirely satisfactory for the preliminary
work. Portions of the above specimens were subjected to treat-
ment with 5 per cent sulphuric acid in 95 per cent alcohol to test
the permanence of the stains. Some decolorization (not decal-
cification) was noted when the time in the 5 per cent acid was ex-
tended over forty-eight hours. This permanence t o sulphuric
acid applies to the skeleton of the chick a t hatching and to the
 DIFFERENTIAL STAINING OF BONE 163

skeletons of pig embryos of the stages mentioned. Sulphuric acid

has not been satisfactory when used on the bone of young rats.
Lundval ( ’05) must have had in mind the acids that form soluble
calcium salts when he comments, “ Da das Alizarin in sauren Lo-
sungen beinahe vollstandig entfiirbt wird, kann man keine von
den schon beschriebenden Knorpelfarbenmethoden verwenden.”
Calcium sulphate is quite insoluble. I t should be noted here that
in differentiating specimens stained by sodium alizarin mono-
sulphonate, the acidulation of the alcohol with sulphuric acid
causes the red soft tissues to become immediately yellow, while the
bone remains red. This yellow color is readily extractable.
Spalteholz (’14) made use of these facts in his earlier technic by
adding small amounts of acetic acid to the staining solution itself.
SU MMART

Of these new bone stains indigo-carmine, Rordeaux red, ali-

.zarol black 3G (National Analine and Cheniical Co.) and alizarine
black SBB powder (Bayer), seem particularly suitable for stain-
ing preserved specimens.
The number of stains shown to combine differentially with bone
indicates that there is a large number of mordant or adjecttive
dyes with this property.
The anthraquinone grouping is not necessary for bone staining.
Weak solutions of sulphuric acid are adequate and satisfactory
decolorizing agents for soft tissues in differentiating bone stains.
Staining of bone in preserved specimens is largely a matter of
differential decolorization.

I wish t o thank Dr. A. G . Pohlman, of St. Louis University, in

whose laboratory the preliminary experiments for this paper were
carried out, and t o acknowledge especially my indebtedness t o
Prof. R. Fischer, of the Department of Chemistry of this Uni-
versity, who placed a collection of alizarines at my disposal.
164 OSCAR V. BATSON

BIBLIOGRAPHY

BELCHIER,JOHN 1736 An account of the bones of animals being changed t o a

red colour by aliment only. Philosophical Transactions. Royal
Society of London, 1735-1736, vol., 39, p. 287.
BROOKS,B. 1917 Studies in regeneration and growth of bone. Annals of
Surgery, June, p. 705.
1920 Studies in bone transplantations. Archives of Surgery, vol.
1, p. 284.
EHRLICH,P. 1885 Das Sauerstoff Bedurfniss des Organismus. Berlin: Verlag
von August HirshwoId.
GOTTLIEB,B. 1914 Die vitale Fiirbung der kalkhaltigen Gewebe. Anat. Anz.
Bd, 46, S. 179.
HILL,EBEN C. 1906 On the Schultze clearing method as used in the Anatomical
Laboratory of the Johns Hopkins University. J. 13. Hosp. Bid., vol.
17, p. 111.
LEMNIUS, LAEVINIUS1581 De miracules Occultis Naturae. Antwerp Ex
Officina Christophori Plantini.
LUNDVALL, HALVAR1W4 Ueber Demonstration embryonaler Knorpelskelette.
. Anat. Anz., Bd. 25, S. 219.
1905 Weiteres uber Demonstration embryonaler Skelette. Anat.
Anz., Bd. 27, S. 521.
MILLER,CHAS.H. 1921 Demonstration of the cartilaginous skeleton in mam-
malian fetuses. Anat. Rec., vol. 20, p. 415.
RAWITZ,BERXHARD1896 Die Venvendung der Alizarinc und Alizarincyanine
in der histiologischen Technik. Anat. h e . , Bd. 11, S. 294.
SPALTEHOLZ, W. 1914 Ueber das Durchsichtigmachen von menschlichen und
tierischen Priiparaten, 2. Auflage. Leipzig: S. Hirzel.