Recent Research in Science and Technology 2011, 3(2): 54-58

ISSN: 2076-5061
www.recent-science.com

MEDICAL SCIENCES

MUSEUM PRESERVATION OF SKELETON OF FETUS & SMALL

VERTEBRATES
K. Leena Pramod1∗, Vina Ravi Vaswani2, Bindhu. S1
1Department of Anatomy, Yenepoya Medical College, Yenepoya University, Deralakatte, Mangalore, Karnataka, India
2Department of Forensic Medicine, Yenepoya Medical College, Yenepoya University, Deralakatte,
Mangalore, Karnataka, India

Abstract
Transparency technique to demonstrate cartilage and skeleton has been used from years. It is superior to the method of
obtaining fetal skeleton by boiling and burying, consisting basically of muscle digestion and staining of cartilage and skeleton.
The process involves maceration of soft tissues in 1% KOH and staining of specimen using Alcian Blue 8GX and Alizarin Red
S allowing detection of morphology of whole vertebral column and single vertebra, long bones and primary ossification centers.
Keywords: Maceration, Alcian Blue, Alizarin Red S, KOH, Primary ossification centre, Fetal skeleton, Small vertebrate
skeleton

Introduction Objectives
Earlier methods of skeleton preparation involved Study the state of cartilage & bones in different
burying of foetus for 2-3 months and slowly recover the stages of development using Alcian blue & Alizarin Red
bones to construct the skeleton or to display loose S. To demonstrate museum specimens skeleton
foetal bones. Foetal remains contain many very tiny preserved in fluid or as dry mount. Preparation of small
bones which have not yet fused with others and are vertebrate skeleton
extremely easily lost into the soil. Another method
involved process of maceration in which the specimen Materials and Method
is boiled in a solvent (water) to remove the last of flesh, We collected fetuses between the age group of 2
grease, and cartilage (3). Sometimes bleaching agents months to 7 months, from Yenepoya Medical college
are added to the water. The method described in this hospital & small adult vertebrates for our study.
article is simple and effective to evaluate bone in small Bone cartilage double stain technique includes
embryos, fetus, small vertebrates. following steps.
The specimen is digested in KOH, stained with Fixation I : Fix specimens immediately in 10%
Alcian Blue 8 GX and Alizarin Red S, cleared in Formalin (12), for a day to a week depending on the
increasing concentration of glycerin, when completed size of the specimen.
the cartilage are blue in color and bones are red in the Evisceration: Remove the skin, eyes, thoracic &
transparent muscle. The specimen can be stored in abdominal viscera, adipose tissue (this step can be
pure glycerin. The principle used here is the affinity of omitted in case of early embryos). (11).
Alizarin Red S to bind with Calcium of bones; this dye Washing: wash the specimens thoroughly in running
stains only the ossified areas of bone. It gives red tap water for 2 days
colour to the bone. The dye was originally made from Fixation II: specimen is fixed in 95% alcohol for 2 to 4
plant madder (Ruba tinctorum), it was first used in 18th days(This step helps skeletons withstand maceration).
century experiments on bone growth by John Hunter Defattening: specimen is put in acetone for 2 to 3
and others (4). Before 8th week of foetal life the bones days(till acetone doesn’t show yellow discolouration.)
of lower limb are composed not of bone, but hyaline Maceration: Keep the specimen in 1% KOH till the
cartilage. They are often called cartilage models. bones are visible through the surrounding tissue.(3)
Cartilage is an essential growth tissue of bones and it Cartilage staining: place specimen in 20mg Alcian Blue
can grow very quickly. The story of bone development in 70% alcohol and 30ml glacial acetic acid for 12 to 24
in the lower limb is story of gradual replacement of hours. The whole specimen turns blue along with the
cartilage by bone tissue, first in the shaft then later at cartilage.
the ends of the cartilage models, as rate of growth Destaining: Place the specimen in 70% alcohol. Keep
declines. changing the alcohol till the solution shows no blue
colouration. (This dehydration will fix the Alcian Blue in

∗
Corresponding Author, Email: leens.pr73@yahoo.co.in
 K. Leena Pramod et al./Rec Res Sci Tech 3 (2011) 54-58

cartilage and help destain surrounding soft tissues.) According to Goldstein and Horobin cartilage stained
(11). dye is removable by Magnesium chloride which we
Bone staining: Place specimen in 0.1% Alizarin Red S have not tried. The specimen should not be kept in
in 1% KOH till the bones are stained red.(3). alcian blue for long, destaining should be done
Clearing: The specimen is placed in a solution immediately in 95% alcohol, with repeated change of
containing KOH-1g, Glycerin-20ml, Water-79ml, till the solution. Cartilage staining is not reversible so careful
excess red colour is removed.(3). observation of specimen is a must. We can skip this
The specimen is placed in 10% glycerin, followed step of cartilage staining and go directly for alizarin
by 50%, 70%, and finally mounted in 100% glycerin. A staining, here only the bone will take up red colour and
few crystals of phenol or thymol can be added. All the cartilage will appear transparent (fig 5 and 6). In case
chemicals used can be obtained from commercial of pigmented specimens bleaching is done. Specimen
biological supply. should not be bleached for long as gas bubbles will
form within the skeleton, change to 1% KOH solution
Discussion immediately if bubbles are formed and repeat changes
The degree of progression of ossification from until no air bubbles are formed, then the specimen is
primary centre, which is endochondral in appendicular subjected to clearing (15) but we found that we get
and axial skeleton, except for clavicle and intra good results if bleaching is done after digestion. Most
membranous centers, the majority of cranial bones are of the earlier published work has suggested clearing of
visible (4). The carpus and talus are wholly soft tissues[digestion] in trypsin ( 12), which is
cartilaginous except for primary centre of calcaneous expensive, we have used 1% KOH for clearing with
as are the epiphyses of long bones. The central and good result.We macerated the specimen in 1% KOH
neural arches of vertebrae are separate. The sternum before cartilage staining in foetus above 6 months
is still unossified. The membranous anterolateral and instead of staining the specimen first and then
postero lateral fontanelles are obvious (fig 4 &7). subjecting it to maceration (11). Our method took less
According to Dawson’s technique (3), the specimens time to destain. Here we would like to add that
are fixed in 95% alcohol directly without first fixing in destaining after cartilage staining is very important as
10% formalin, in the first case maceration of tissues the specimen when put in Alizarin Red S the cartilage
take lesser time when compared to specimens first turns to green colour .When specimens are being
fixed in 10% formalin but are liable to greater degree of cleared they soften and if left too long, macerate and
damage if not handled carefully as specimens are are destroyed. Fixation in 10% formalin hardens the
fragile and not hardened as in formalin fixation. tissues and helps to control the maceration, though it
Staining of cartilages has been done in the past using prolongs the clearing process (2). 95% ethyl alcohol is
methylene blue or toluidene blue (2) but the colour used for fixation or 10% formalin followed by 95%
fades with time. Different procedures have been given alcohol, this makes fragile skeleton hard. It has been
for preparing alcian blue stain for cartilage staining.3% suggested that concentration of KOH can be increased
alcian blue 8GXin glacial acetic acid, alcian blue 8 GN for larger specimens (5), but careful monitoring is very
-10mg, 80ml 95% ethyl alcohol& 20% glacial acetic important lest the specimen will be damaged. In (fig8)
acid (12), 1 vol 0.3% alcian blue 8 GS in 70% the finer bones (carpal bones, tarsal bones, phalanges,
ethylalcohol(5). As suggested by Simons & Van cartilaginous ends etc) have been disarticulated.
Horn(1971) staining of cartilage is now done with Evisceration of specimens hastens the maceration
Alcian blue 8 GX stain. In our study we used 20mg process; small embryos which are already transparent
alcian blue in 70% Absolute Alcohol and 30ml Glacial need not be eviscerated. Skin of frog should be
acetic acid (11), some have used toluidine blue removed first before fixation and the tail of the lizard
instead of alcian blue, the former gives problem of should be slit length wise so that subsequent swelling
decolourisation of cartilage when placed in alkaline will not break the tail off(2). For bone staining we
solution (5).Monitoring of specimen kept in alcian blue placed the specimen in 0.1% Alizarin Red S in 1%
staining is important to prevent overstaining as staining KOH (3) and kept the specimen in clearing solution (1g
cannot be removed from cartilage by any of the KOH, 20ml Glycerin and water 79ml) for a longer time
chemicals used in the clearing and staining process till the solution didn’t show red colour The specimen
(15).Phenol removes alcian blue staining (17), the should be kept on white surface in sunlight to speed up
chemical is highly volatile and produce severe burns if the process (6). For the next clearing step we used
it comes in contact with skin, destaining should be 10%, 50%, 70% & finally 100% of Glycerin instead of
done in a well ventilated area [phenol can cause 10% to 100% glycerin(3, 11). The skeletons which
headaches] , in glass jar, using gloves while handling were over macerated were dry mounted (fig 8). The
the specimen but when we tried to destain our bone cartilage double staining technique reveals even
specimen using phenol we didn’t get the desired result, slight amounts of cartilage and mineralized tissues,
making it valuable in studies of skeletal development in
 K. Leena Pramod et al./Rec Res Sci Tech 3 (2011) 54-58

embryos, fetuses specimens, and small adult Fig-5

vertebrates. We have prepared these to be displayed
in the museum for students to observe the ossification
centers & to study comparative anatomy of skeleton
displayed.

Fig-1

Fig-6

Fig-7

Fig-2

Fig-3 Fig-8

Fig-4
 K. Leena Pramod et al./Rec Res Sci Tech 3 (2011) 54-58

Fig-9 interest, it is important that the specimen is not placed

in fixation until the skin is removed.
In the early stained skull there are several obvious
fontanels, the single frontal fontanel on the top,
between frontal and parietal bones,(fig 4) bilateral
sphenoid fontanel above eye and jaws, bilateral
mastoid fontanel behind ears, single occipital fontanel
at the back of the skull ( fig 7).These sutures can be
easily identified from rest of the bones because they
are more triangular instead of narrow elongated spaces.
Fig-10 The roof and sides of the skull form as membrane
bones without formation of cartilage first, where as the
base and palate form cartilage and then bone. The
fontanels are in membrane bones. There are similarity
between all the vertebrae with a vertebral canal and
neural arch which surrounds it. Only small dots of
bones are present in the stained specimen (fig 7). As
per our study by using alizarin Red S and alcian blue
it is easy to demonstrate the nature of appearance of
ossification centers in fetus and small animals, and
cartilaginous skeleton can be prepared without
Fig-11
distortion from shrinkage.

Acknowledgements
My sincere thanks to Mr. Hashim. A, Toxicologist,
Ms. Bhavani, Technician and Mr. Kiran, Modeller for
helping me to carry out this study.

References
1. Potthoff, T. 1984. Clearing and staining
Fig-12 techniques. pp 35-37 in: H.G.Moser, W.J.
Richards, D.M. Cohen, M.P. Fahay, A.W. Kendall
Jr. and S.L. Richardson, eds. Ontogeny and
Systematics of Fishes. American Society of
Ichthyologists and Herpetologists. Special
publication No.1.
2. Milton Hildebrand. A hand book of Anatomic
preparations. University of California Press. 1968;
(p 17-30, 43-50).
Fig-13 3. C F A Culling. Handbook of histopathological
techniques (including museum technique).
London, Butterworths, 1963 [ p-523-538]
4. Gray’s Anatomy: The Anatomical Basis of Clinical
Practice, Publisher: Churchill Livingstone 38th Ed,
1995; [p 471-479]
5. Double staining technique for rat foetal skeleton in
teratological studies. J Whitaker & Kathleen. M.
There are some permanent cartilage that do not Dix Davis . D. Dwight and Ulys Roy Gore. Field
ossify such as cartilage of trachea and articular Mus. Of Natur. Hist. Technique series No. 4
cartilage of joints, intramembranous bone formation October 1936 P.1
occur directly without a cartilage template.(4) When 6. Alcian blue staining of the mouse foetal
preparing double stained specimens, the overlying skin cartilaginous skeleton. Andras Nagy, Marina
of the specimen must be removed to allow Alcian blue Gertsentein, Kristina Vinterstein, Richard
to penetrate and stain the cartilage (13), the skin is Behringer. CSHL Press, Cold Spring Habor,
difficult to remove from the extremities of fixed Ny, USA 2003
specimens so if cartilage formation in digits is of
 K. Leena Pramod et al./Rec Res Sci Tech 3 (2011) 54-58

7. O’rahilly, &Gardner E. The initial appearance of adult bats and rodents. Int. J. Morphol, 27(4);
Ossification in staged human embryos. Am J 1165-1167 2009
Anat.1972; 134: 291-308 12. Narotsky M.G, Rogers. JM. Examination of axial
8. Large scale double staining of rat foetal skeletons skeleton of fetal rodents. Developmental biology
using Alizarin Red S & Alcian blue. Angela.D protocol Vol.1; 139-141
Young, Donal E.Phipps, Barry Astroff. Teratology, 13. Goldstein D. J., R.W. Horobin. Rate factors in
6(4); 273—276 staining by alcian blue. Histochem. J.,1974; 6,
9. Simons E.V.,and J.R.Van Horn. 1971. A new 157-174.
procedure for whole mount alcian blue staining of 14. Diagnostic - staining protocol of cartilage and
the cartilaginous skeleton of chicken embryos, bone, Synnove Helland Wilson Bull, Technique for
adapted to the clearing procedure in potassium avian syringes June 1988,100(2); 289-293
hydroxide. Acta Morphol. Neerlscand,8:281-292 15. Taylor, W. R. & G. C. Van Dyke. 1985. Revised
10. Functional photography, july/Aug 1981, vol 16, no. procedures for staining and clearing small fishes
4, chapter 22,The visible skeleton. Dr. James and other vertebrates for bone cartilage study.
Hanken , Dr. Richard Wassersug. Cybium9(2):107-119.
11. Cortes – Delgado.N; Perez-Torres J.R Hoyos J.M.
Staining procedure of cartilage and skeleton in