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Technique Series

Field Museum op Natural History

Founded b\ Marshall Field, 1893

Number 4

CLEARING AND STAINING SKELETONS
OF SMALL VERTEBRATES

BY

D. DwiGHT Davis

ASSISTANT, DIVISION OF OSTEOLOGY
AND

U. R. Gore

Wilfred H. Osgood

CiniATOB, DEPARTMENT OF ZOOLOGY
EDITOR

CHICAGO, U.S.A.
October 31, 1936

Technique Series

Field Museum of NatuPwAL History

Founded by Marshall Field, 1893

Number 4

CLEARING AND STAINING SKELETONS
OF SMALL VERTEBRATES

BY

D. DwiGHT Davis

ASSISTANT, DIVISION OF OSTEOLOGY
AND

U. R. Gore

THE LIBRA'^Y OF THE

_ NOV 16 1936

UNlVERSiTY OF iLLiNOlS

Wilfred H. Osgood

CURATOR, DEPARTMENT OF ZOOLOGY
EDITOR

CHICAGO, U.S.A.
October 31, 1936

PRINTED IN THE UNITED STATES OF AMERICA
BY FIELD MUSEUM PRESS

J / /

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CLEARING AND STAINING SKELETONS
OF SMALL VERTEBRATES

BY D. DWIGHT DAVIS AND U. R. GORE

The preparation of osteological material has always been a
source of annoyance to anatomists. With any of the smaller forms,
from fishes to mammals, satisfactory results demand an expenditure
of time out of proportion to the results obtained. A delicate skeleton,
no matter how carefully handled, inevitably suffers more or less
damage from continued use. Furthermore, with most small fishes
and many amphibians and reptiles, even an expert finds it next to
impossible to produce really satisfactory results. The abundant
cartilage found in many forms, which dries into an extremely friable
and almost shapeless mass, further complicates matters. In addition,
since warping and distortion of slender or thin bones and calcified
cartilage may make the exact form and relation of parts almost as
much a matter of conjecture as of observation, work on the smaller
vertebrates has been so impeded that the extremely interesting
comparative osteology of these animals is still almost unknown.

' The exclusive use of external characters in defining the major
groups of the lower vertebrates is no longer practical. The extraor-
dinary success which has been achieved in recent years by comparing
the internal anatomy of these forms is perhaps an indication of
what may be anticipated along these lines. Unfortunately, accurate
results in this type of work demand an examination of extremely
large series of specimens. In addition, the rapid rise of paleontology

j. has given comparative osteology a new lease on life, and has at
the same time made the imperfect state of existing knowledge all
too apparent.

Cleared specimens cannot, of course, entirely supplant dry
skeletons even of the smaller forms, except in certain types of work.

. Rather the cleared specimens should be viewed as supplementary ones.

f Some studies demand more or less completely disarticulated skeletons.

- Certain features of the skeleton can be observed with difficulty or not
at all when the bones are surrounded by a layer of flesh, however
transparent the latter may be. This is particularly true of the skull,
where the general complexity of structure, the thickness of bony

5 3

4 Field Museum of Natural History— Technique, No. 4

masses, and the presence of such dense masses of flesh as the eyes'
and the tongue make accurate observation of details quite im-
possible. For the post-cranial skeleton, however, cleared material
may be employed to great advantage.

A list of the uses of the modified Schultze method could hardly
be complete, but some of the more important applications may be
pointed out. Unquestionably its greatest advantage lies in extraor-
dinary saving of time. In the hands of a single careful technician,
unlimited series of uniformly good specimens may be prepared in
a surprisingly short time. A further general advantage is the fact
that a skeleton is always complete. No small bones or delicate proc-
esses are lost during preparation, and none become detached and
lost from careless handling later on. Glycerine-preserved specimens
may also be manipulated to a certain extent, and actual dissection
of parts may easily be carried out when necessary. More specifically, a
host of special uses comes to mind. The preservation of cartilaginous
parts in their normal condition has already been alluded to. The
same applies to calcified cartilage, which is, if anything, even
more brittle when dry. The entire pectoral girdles of amphibians
and reptiles, the sternal and abdominal ribs of the latter, small
isolated nodules of calcified cartilage (e.g. the hypoischium of lizards),
may be examined in a complete and undisturbed condition. The
small but highly important bones of the carpus and tarsus are not
obscured by masses of dried ligaments, which unfortunately must
be retained as a binder in dried preparations. The method has
advantages when applied to the study of the vestiges of limbs and limb
girdles in degenerate types, as anyone who has tried to dissect out
these parts can testif}^ Equally obvious are its uses in the study of
larval skeletons, and of the time and places of ossification in the ontog-
eny of individual bones. The intricate structure of the axial skeleton
in the higher teleost fishes, with their complexities of extremely delicate
rib and vertebral structure, is rendered beautifully apparent.

The application of this method to museum exhibition is almost
a virgin field. Good preparations are by no means unattractive, and
could be profitably introduced as a supplement to dried mounts.
The curious question, so often encountered, "What do you make
the bones of?" probably would not be asked on viewing an exhibit of
this type. While the uses of the technique in this field are limited,
they are none the less interesting, and could be explored to advantage.

' Of course the eyes may be removed. In reptiles, however, this destroys
the sclerotic rings, which are an integral part of the skeleton. It is impossible
to remove the tongue without damaging the hyoid apparatus.

Fig. 1. Horned lizard, Phrynosoma cornutum, photographed from the ventral side. Prepared
from a freshly-killed specimen.

6 Field Museum of Natural History— Technique, No. 4

HISTORICAL SUMMARY

The use of various agents for clearing animal tissues is by no
means new. Until recently this technique, usually in combination
with bone stains or injections of blood vessels, has been almost
exclusively employed in studies on human embryos and a few
standard laboratory types. The possibility of utilizing it in studying
the skeletons of various small vertebrates, where the preparation
of dry skeletons is tedious and unsatisfactory, has only recently been
exploited.

The several processes now in use are all adaptations of one of two
original techniques. The SpaUeholz method, which is essentially a
modification of standard histological procedure, is open to several
objections when applied to skeletons, and is no longer in general use.
The most serious disadvantage, in addition to the time involved
and the expense of some of the materials used, is the impermanence
of the specimens, which discolor rapidly unless they are mounted
in balsam. Schultze (1897) first formulated the technique of using
potash and glycerine in clearing human embryos, and any technique
involving clearing with potassium hydroxide is generally referred
to as the Schultze method. He used KOH solutions as strong
as 20 per cent, but subsequent investigation has reduced this
concentration considerably. Later, Mall (1906) introduced the
technique into this country, where it has been widely used, in more
or less modified forms, in studies on ossification. An important
addition to the original Schultze technique was made by Lundvall
(1905), who introduced the use of alizarin for staining the bones.
Although various stains have been used, alizarin has been almost
universally adopted because of its selective properties.

The Schultze process is essentially one of rendering all the fleshy
parts of an animal more or less transparent by means of a strong
alkaline solution and glycerine and by removing pigment with hydro-
gen peroxide, ultra-violet light, or other bleaching agents. At the
same time the calcified sti-uctures are brought into sharp relief by
staining them with a selective dye.

Thomas Barbour has published evidence which indicates that
he was the first to employ this process on small vertebrates. The
figure of SminthUlus limbatus that appears on Plate I of a paper
by him and Noble (1920) bears the caption "Specimen cleared after
Schultze's method by Thomas Barbour in 1913." Noble (1917)
used a modification of the technique on the toes of frogs and in his
"Herpetologj- of the Belgian Congo" (1924) figures a series of pectoral

Clearing Small Vertebrates — Davis and Gore 7

girdles prepared by this method. More recently Beebe (1929),
and particularly his assistant, Hollister (1932, 1934), have used it
on a mass production basis on fishes. Although Hollister (1934)
has given an outline of the process she employs on fresh fishes, an
adequate and detailed discussion of the method with reference to
the various vertebrate groups has long been needed, especially with
reference to preserved material, and its lack has doubtless impeded
the wide application that the process merits.

The present study was undertaken in an effort to fill the need for a
simple, reasonably rapid method of making the large series of good
osteological preparations so necessary in various types of taxonomic
and anatomical studies. An important consideration in this con-
nection is the fact that studies of this nature can rarely, if ever, be
made on fresh material. Museum specimens, which are used in
most investigations of this type, are universally preserved in 60-65
per cent alcohol. This type of material has been used in this study,
together with enough fresh material to serve as a basis for comparison.

While preserved material rarely yields the crystal-clear prep-
arations that may be made with fresh specimens, this objection is
by no means a serious one, since in most instances the results are
sufficiently transparent to photograph well.

MATERIALS AND METHODS

In view of Hollister's account of the treatment of fishes, our work
centered largely on amphibians and reptiles, the only other vertebrate
groups that are habitually preserved in toto. It included, however,
a number of fishes and several nestling birds and mammalian embryos.
An interesting possibility was opened up by the unusual success
achieved with a small bat, a form in which the delicate skeleton is
exceedingly difficult to prepare in the orthodox way. A wide selec-
tion of sizes and degrees of ossification was made among the sala-
manders, frogs, and lizards.

On general principles, a good grade of chemicals was used in
making up the solutions. C.P. potassium hydroxide sticks are
preferable for making the KOH solutions. Distilled water was used
throughout as a precautionary measure. According to Hollister
its use is especially desirable in making up the stain, since flocculent
suspensions may result if tap water is used. A stock solution of 10 per
cent KOH was prepared, and the lower percentages were made up
from it as necessary. U.S. P. white glycerine was used, with excel-

8 Field Museum of Natural History— Technique, No. 4

lent results. The stock dye was made up according to the formula
recommended by Hollister:

Alizarin, sat. sol., in: cc.

Glacial acetic acid 5

Glycerine, white 10

Chloral hydrate, c.p., 1 per cent sol 60

Hollister claims that the staining qualities of alizarin made up
according to this formula are superior to the alcoholic alizarin stain
recommended by others. She also advises the addition of the
acetic acid on the grounds that it has a tendency to counteract the
absorption of the stain by the softer tissues and facilitates final
clearing. These points were not checked by us. Her formula gave
uniformly satisfactory results, and was used throughout this work.
As needed, this stock dye solution was added to 4 per cent KOH
solution in the proportions of 1 part of the dye to 1,000 of the KOH.

PROCEDURE

The process falls naturally into four parts, and in order to simplify
the discussion as much as possible each part is taken up step by step,
with a discussion of points of special interest. A brief summary of
the entire procedure is then inserted for easy reference. It is believed
that this method of presentation, if followed carefully, will enable
anyone to produce satisfactory results with a minimum of failures.
Unless otherwise stated, statements in the following pages refer to
preserved material.

INITIAL TREATMENT

Fresh material. — Fresh fish require fixing and hardening in alcohol
for several days before they are started through the clearing process.
For this purpose, 95 per cent alcohol hardens quickly and is generally
more satisfactory than lower percentages. Any shrinkage or dis-
tortion resulting from this concentration seems to be temporary,
since specimens assume their normal proportions during the clear-
ing process. In spite of HoUister's excellent results with fresh fish
without any preliminary fixing, all our specimens so treated macerated
within a short time in 1 per cent KOH, and had to be discarded.
Addition of alcohol to the KOH which she recommends failed to stop
maceration. Hardening of fresh frogs, lizards, and snakes was
also found to be desirable. All material should be carefully and
completely eviscerated.

Preserved material. — Evisceration of all material is highly desir-
able, since otherwise details of the girdles and axial skeleton are
always more or less obscured. Where the digestive tract contains

Fig. 2. Above: Small African ranid frog, Phrynobatrachus francisci, photographed from the
dorsal side (from preserved specimen). Below: Cricket frog, Acris gryllus, photographed from
the ventral side (from fresh specimen).

10 Field Museum of Natural History— Technique, No. 4

much undigested matter it is imperative, and with material that is
to be photographed it is always necessary. Abdominal ribs are
present in many lizards, and the incision and subsequent removal
of the viscera must be done with great care to avoid damaging them.
Where these structures are well developed, it is almost impossible
to avoid injuring them. Our procedure in these cases was to make
a clean cut with a pair of scissors to one side of the ventral midline.
While this severs all the abdominal ribs on one side, the cut is clean
and uniform, and the region of the midline as well as the ribs on the
opposite side are left intact.

The stamped tags of treated paper in current use at Field Museum
(Schmidt, 1932) were found to be ideally suited to marking speci-
mens, both the tags and the linen thread used to tie them going
through the entire process without serious damage. Stamped metal
tags may also be used, since none of the reagents contain corrosive
agents, but are less desirable because of their weight and sharp edges.

After evisceration, specimens are soaked in water over night or
longer, to remove the alcohol. Skinning, although it speeds up the
process considerably, is not desirable, since it is apt to leave the
specimens in a flabby and extremely fragile condition. In such lizards
as skinks, where the skin contains a heavy armor of osteoderms, it is
necessary, however, since the osteoderms stain heavily and obscure
all structures lying beneath them. The scales of many fish also
stain to a greater or less degree, but in these cases removal of the scales
rather than skinning is recommended. This is more easily done
after treatment with KOH,

It must be remembered that formaldehyde is a strong decalcifier
of bone. Material that has been preserved in it for any length of
time is not likely to yield good preparations.

Dried spemnens. — Air-dried specimens were put directly into
1 per cent KOH without any preliminary treatment, and have yielded
some surprisingly excellent preparations. A desiccated moonfish
produced one of the most transparent specimens in the entire series.
The general use of dried material can not, of course, be recommended.
There is nothing to indicate that the specimens so treated are in any
way superior to those made from preserved material. In addition
there is always danger of serious maceration.

CLEARING

Specimens previously preserved in alcohol are put into a bath of
3-4 per cent KOH. Of all the strengths of potash tested, this

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resulted in the best and quickest clearing with the least maceration
of tissues in alcoholic material. On the other hand, a 1 per cent
solution is sufficiently strong for fresh material, and higher con-
centrations are likely to result in maceration. After 24 to 48 hours
in the clearing agent, heavily pigmented specimens are transferred
to full strength hydrogen peroxide until thoroughly bleached.
Bleaching usually takes several hours. The use of peroxide after
KOH treatment rather than before is advantageous in that good
penetration of the peroxide is insured and more thorough bleaching
obtained. The principal objection to the use of peroxide is its tend-
ency to produce air bubbles in the flesh of the specimen. To remove
these one by one with a needle requires time and patience. Actually,
unless they are extremely large and numerous, they do not interfere
to any great degree with the utility of the specimens, except where
material is to be photographed. Treatment with KOH before
bleaching seems to counteract the formation of bubbles to a consider-
able extent.

With the exception of the time in peroxide, the specimens are
exposed to full sunlight in shallow, white, porcelain-lined photo-
graphic trays. Average sunlight is sufficiently strong to make good
transparent preparations with three or four weeks' exposure, and in
even shorter times for some material.

After bleaching, the material is returned to 4 per cent KOH,
where it remains until it is quite transparent. The bones of the toes,
the pectoral girdle, and other parts directly under the surface should
be readily visible, although deeper parts may still be obscure. The
time required for this clearing varies from a day for small frogs
to a week or more for fair-sized lizards.

Greasy bones will not take up the dye. Dehydration and treat-
ment with acetone has been recommended, but in our experience
this was not necessary. Alcohol is a fair degreasing agent, and
specimens that have been preserved in it for some time are usually
fairly free from grease. What little may remain seems to be removed
by the KOH.

Beebe and Hollister used an alpine sun lamp in place of sunlight,
with good results. Its advantages, particularly during cloudy
weather, are obvious. Hollister claims that the use of ultra-violet
light makes the use of bleaching agents for depigmentation un-
necessary. We were unable to test its efficiency on preserved
material.

Clearing Small Vertebrates— Davis and Gore 13

STAINING

The most satisfactory dye solution for preserved specimens
was made with 10 cc. of the stock dye solution (p. 8) per liter of
4 per cent KOH. Material is transferred directly to this solution from
the KOH bath. The time required for staining varies somewhat,
but with a solution of this strength twenty-four hours is sufficient
for most specimens. The flesh takes up a good deal of stain if
specimens are allowed to remain in the dye too long. Although
most of this stain eventually comes away, it makes subsequent
clearing needlessly long and complex, without adding anything to
the usefulness of the preparations. In many instances the stained
flesh is extraordinarily resistant to clearing. On the other hand,
immersion in the dye for too short a time results in a light stain which
does not differentiate satisfactorily. Hollister recommends a 1-1000
alizarin solution in 1 per cent KOH for staining fish, and Dawson
used the same formula of alcoholic alizarin on legs of rats with
good results. This concentration is not strong enough for preserved
amphibians and reptiles since specimens immersed for several days
in 1 per cent KOH, 1-1000, were stained an unsatisfactory light red.

FINAL CLEARING

Material is transferred from the stain to a 4 per cent KOH
solution where it remains twenty-four hours. By this time the
solution is usually deeply colored from the excess stain that has come
away from the specimen, and transfer to a fresh solution is necessary.
This process should be repeated as long as the KOH discolors.
After the material is free from excess stain and fairly transparent, it
may be transferred through the following solutions to pure glycerine:

KOH

Glycerine 4 per cent HOH

(Parts) (Parts) (Parts)

20 3 77

50 3 47

75 0 25

100 0 0

Transfer of material directly into pure glycerine causes great
shrinkage and distortion.

Exposure to sunlight in white-lined trays should continue all
along. Even in pure glycerine it is beneficial in final clearing. A
specimen that is not wholly transparent frequently becomes remark-
ably clear in one or two days when finally placed in pure glycerine.
If specimens are not sufficiently transparent at the end of the treat-
ment, they may, of course, be returned to KOH for further treatment.

14 Field Museum of Natural History— Technique, No. 4

STORING

Specimens should be stored in chemically pure, white glycerine.
Glass-stoppered bottles are advisable. If the glycerine shows signs
of discoloration at any time, it should be replaced immediately, since
the discoloration is rapidly taken up by the specimen. Under any
circumstances this impairs their transparency, and in extreme
instances may render them useless. Under no circumstances should
cork be used, since it inevitably discolors the glycerine, and this
discoloration is taken up by the specimen. A thymol crystal should
be added to each jar to prevent the growth of molds, which is
frequently very rapid and destructive. Material should be stored
away from light, although Hollister claims she has left specimens in
the light for ten years without any signs of deterioration.

SUMMARY OF TREATMENTS

(1) Fresh specimens are hardened for about a week in strong
alcohol (95 per cent). Material preserved in alcohol is washed in
water over night or longer. All material is completely eviscerated.

(2) Transfer fresh specimens to 1 per cent KOH in distilled
water; use 4 per cent KOH for preserved material and expose to
strong sunlight in shallow, white-lined trays for two days.

(3) Bleach thoroughly in full strength hydrogen peroxide.

(4) Return to 4 per cent KOH. Expose to sunlight until
specimen is transparent and bones are easily visible through the skin.

(5) Stain twenty-four hours or longer in 10 cc. stock dye solution
to 1,000 cc. 4 per cent KOH.

(6) Return to KOH (1 per cent or 4 per cent, as stated above),
exposing to sunlight and changing solution until stain ceases to come
away and specimen is transparent.

(7) Transfer gradually to pure glycerine.

(8) Store in glass-stoppered bottles, away from light. Add a
crystal of thymol to the glycerine.

It is important to remember that each specimen presents its own
individual problems. Variations in type of preservation, in the length
of time the material has been stored in alcohol, and in the size
and consistency of the specimen itself, are such that any but the
most general rules would be valueless and misleading. Each speci-
men demands careful watching in all stages of the process, and
unquestionably the factor of personal experience on the part of the
technician is an important one in producing results that are uniform

Clearing Small Vertebrates — Davis and Gore 15

in quality. Some idea of the variations encountered may be gained
from the following notes:

An adult, freshly killed horned toad {Phrynosoma) was cleared in
four days in 3 per cent KOH, was stained in twenty-four hours, and
was quite transparent in two weeks' time (fig. 1).

A series of fresh cricket frogs {Acris gryllus) was cleared and
stained in a week, resulting in almost crystal-clear preparations (fig. 2).

Preserved specimens of the larger ranids (Rana pipiens, R.
clamitans) required about three weeks for the entire process.

Fresh lizards (Eumeces, Crotaphytus) were ready for study in
three weeks. A series of twenty preserved iguanid lizards were
cleared in from two to five days, stained in twenty-four hours, and
cleared satisfactorily in from one to two months' time. THE liBR'"^^ OF the

REFERENCES ^^' U s/ 1 6 1936

Barbour, T. and Noble, G. K. "fMtVf RSITY of illinoi?

1920. Some Amphibians from Northwestern Peru, with a Revision of the Genera
Phyllobates and Telmaiobius. Bull. Mus. Comp. Zool., 63, pp. 395-427.

Beebe, W.

1929. The Architectural Beauty of Fish. New adaptations of an old method.
Bull. N. Y. Zool. Soc, 32, pp. 67-69.

Dawson, A. B.

1926. A Note on the Staining of the Skeleton of Cleared Specimens with
Alizarin Red S. Stain Technol., 1.

Hill, E. C.

1906. On the Schultze Clearing Method as Used in the Laboratories of the
Johns Hopkins University. Bull. Johns Hopkins Hosp., 17, pp. 111-115.

HOLLISTER, G.

1932. A New Use of Ultra-Violet Radiation. Bull. N. Y. Zool. Soc, 35,

pp. 49-50.
1934. Clearing and Dyeing Fish for Bone Study. Zoologica, 12, pp. 89-101.

LUNDVALL, H. VON

1905. Weiteres iiber Demonstration embryonaler Skelette. Anat. Anz., 27,
pp. 520-525.

Mall, F. P.

1906. On Ossification Centers in Human Embryos Less than One Hundred
Days Old. Amer. Journ. Anat., 5, pp. 433-458.

Noble, G. K.

1917. The Systematic Status of Some Batrachians from South America.

Bull. Amer. Mus. Nat. Hist., 37, pp. 797-814 (p. 795).
1924. Contributions to the Herpetology of the Belgian Congo Based on the

Collection of the American Museum Congo Expedition, 1909-1915. Bull.

Amer. Mus. Nat. Hist., 49, pp. 147-347.

Schmidt, K. P.

1932. Museum Tags of Chemical Proof Paper. Science, 75, pp. 23-24.
Schultze, O.

1897. Ueber Herstellung und Conservirung durchsichtiger Embryonen zum

Stadium der Skeletbildung. Anat. Anz., 13.
1897. Grundriss der Entwicklungsgeschichte des Menchen und der Sauge-
thiere. p. 459.

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