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 vertebrat

Vina Ravi Vaswani2, Bindhu. S1, K. Leena Pramod1,

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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                                                          ∗ Corresponding Author, Email: leens.pr73@yahoo.co.in Introduction Earlier methods of skeleton preparation involved burying of foetus for 2-3 months and slowly recover the bones to construct the skeleton or to display loose foetal bones. Foetal remains contain many very tiny bones which have  not yet fused with others and are extremely easily lost into the soil. Another method involved process of maceration in which the specimen is boiled in a solvent (water) to remove the last of flesh, grease, and cartilage (3). Sometimes bleaching agents are added to the water. The method described in this article is simple and effective to evaluate bone in small embryos, fetus, small vertebrates. The specimen is digested in KOH, stained with Alcian Blue 8 GX and Alizarin Red S, cleared in increasing concentration of glycerin, when completed the cartilage are blue in color and bones are red in the transparent muscle. The specimen can be stored in pure glycerin. The principle used here is the affinity of Alizarin Red S to bind with Calcium of bones; this dye stains only the ossified areas of bone. It gives red colour to the bone. The dye was originally made from plant madder (Ruba tinctorum),  it was first used in 18th century experiments on bone growth by John Hunter and others (4).  Before 8th week of foetal life the bones of lower limb are composed not of bone, but hyaline cartilage. They are often called cartilage models. Cartilage is an essential growth tissue of bones and it can grow very quickly. The story of bone development in the lower limb is story of gradual replacement of cartilage by bone tissue, first in the shaft  then later at the ends of the cartilage models, as rate of growth  declines.   Objectives Study the state of cartilage & bones in different stages of development using Alcian blue & Alizarin Red S. To demonstrate museum specimens skeleton preserved in fluid or as dry mount. Preparation of small vertebrate skeleton  Materials and Method We collected fetuses between the age group of 2 months to 7 months, from Yenepoya Medical college hospital & small adult vertebrates for our study.   Bone cartilage double stain technique includes following steps.  Fixation I :  Fix  specimens  immediately in 10% Formalin (12), for a day to a week depending on the size of the specimen.  Evisceration: Remove the skin, eyes, thoracic & abdominal viscera, adipose tissue (this step can be omitted in case of early embryos). (11). Washing: wash the specimens thoroughly in running tap water for 2 days Fixation II: specimen is fixed in 95% alcohol for 2 to 4 days(This step helps skeletons withstand  maceration). Defattening: specimen is put in acetone for 2 to 3 days(till acetone doesn’t show yellow discolouration.) Maceration:  Keep the specimen in 1% KOH till the bones are visible through the surrounding tissue.(3) Cartilage staining: place specimen in 20mg Alcian Blue in 70% alcohol and 30ml glacial acetic acid for 12 to 24  hours. The whole specimen turns blue along with the cartilage. Destaining: Place the specimen in 70% alcohol. Keep changing the alcohol till the solution shows no blue colouration. (This dehydration will fix the Alcian Blue in K. Leena Pramod et al./Rec Res Sci Tech 3 (2011) 54-58  cartilage and help destain surrounding soft tissues.) (11). Bone staining: Place specimen in 0.1% Alizarin Red S in 1% KOH till the bones are stained red.(3). Clearing: The specimen is placed in a solution containing  KOH-1g, Glycerin-20ml, Water-79ml, till the excess red colour is removed.(3). The specimen is placed in 10% glycerin, followed by 50%, 70%, and finally mounted in 100% glycerin.  A few crystals of phenol or thymol can be added.  All the chemicals used can be obtained from commercial biological supply.   Discussion  The degree of progression of ossification from primary centre, which is endochondral in appendicular and axial skeleton, except for clavicle and intra membranous centers, the majority of cranial bones are visible (4).   The carpus and talus are wholly cartilaginous except for primary centre of calcaneous as are the epiphyses of long bones. The central and neural arches of vertebrae are separate.  The sternum is still unossified. The membranous anterolateral and postero lateral fontanelles are obvious (fig 4 &7).  According to Dawson’s technique (3), the specimens are fixed in 95% alcohol directly without first fixing in 10% formalin, in the first case maceration of tissues take lesser time when compared to specimens first fixed in 10% formalin but are liable to greater degree of damage if not handled carefully as specimens are fragile and not hardened as in formalin fixation. Staining of cartilages has been done in the past using methylene blue or toluidene blue (2) but the colour fades with time. Different procedures have been given for preparing alcian blue stain for cartilage staining.3% alcian blue 8GXin glacial acetic acid,  alcian blue 8 GN -10mg, 80ml 95% ethyl alcohol& 20% glacial acetic acid (12), 1 vol 0.3% alcian blue 8 GS in 70% ethylalcohol(5).  As suggested by Simons & Van Horn(1971) staining of cartilage is now done with Alcian blue 8 GX stain.  In our study we used 20mg alcian blue in 70% Absolute Alcohol and 30ml Glacial acetic acid  (11), some have  used toluidine blue instead of alcian blue, the former gives problem of decolourisation of cartilage when placed in alkaline solution (5).Monitoring of specimen kept in alcian blue staining is important to prevent overstaining as staining cannot be removed from cartilage by any of the chemicals used in the clearing and staining process (15).Phenol removes alcian blue staining (17), the chemical is highly volatile and produce severe burns if it comes in contact with skin, destaining should be done in a well ventilated area [phenol can cause headaches] , in glass jar, using gloves while handling the specimen but when we tried to destain our specimen using phenol we didn’t get the desired result, According to Goldstein and Horobin cartilage stained dye is removable by Magnesium chloride which we have not tried. The specimen should not be kept in alcian blue for long, destaining should be done immediately in 95% alcohol, with repeated change of solution. Cartilage staining is not reversible so careful observation of specimen is a must. We can skip this step of cartilage staining and go directly for alizarin staining, here only the bone will take up red colour and cartilage will appear transparent (fig 5 and 6).  In case of pigmented specimens bleaching is done. Specimen should not be bleached for long as gas bubbles will form within the skeleton, change to 1% KOH solution immediately if bubbles are formed and repeat changes until no air bubbles are formed, then the specimen is subjected to clearing (15) but we found that we get good results if bleaching is done after digestion. Most of the earlier published work has suggested clearing of soft tissues[digestion] in trypsin ( 12), which is expensive, we have used 1% KOH for clearing with good result.We macerated the specimen in 1% KOH before cartilage staining in foetus above 6 months instead of staining the specimen  first and then subjecting it to maceration (11). Our method took less time to destain. Here we would like to add that destaining after cartilage staining  is very important as the specimen when put in Alizarin Red S the cartilage turns to green colour .When specimens are being cleared  they soften and if left too long, macerate and are destroyed. Fixation in 10% formalin hardens the tissues and helps to control the maceration, though it prolongs the clearing process (2). 95% ethyl alcohol  is used for fixation or 10% formalin followed by 95% alcohol,  this makes fragile skeleton hard. It has been suggested that concentration of KOH can be increased for larger specimens (5), but careful monitoring is very important lest the specimen will be damaged. In (fig8) the finer bones (carpal bones, tarsal bones, phalanges, cartilaginous ends etc) have been disarticulated. Evisceration of specimens hastens the maceration process; small embryos which are already transparent need not be eviscerated. Skin of frog should be removed first before fixation and the tail of the lizard should be slit length wise so that subsequent swelling will not break the tail off(2).  For bone staining we placed the specimen in 0.1% Alizarin Red S in  1% KOH (3) and kept the specimen in clearing solution (1g KOH, 20ml Glycerin and water 79ml) for a longer time till the solution didn’t show red colour The specimen should be kept on white surface in sunlight to speed up the process (6).   For the next clearing step we used 10%, 50%, 70% & finally 100% of Glycerin instead of 10% to 100% glycerin(3, 11). The skeletons which were over macerated were dry mounted (fig 8).  The bone cartilage double staining technique reveals even 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 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-2           Fig-3        Fig-4     Fig-5        Fig-6         Fig-7              Fig-8             K. Leena Pramod et al./Rec Res Sci Tech 3 (2011) 54-58  Fig-9       Fig-10       Fig-11       Fig-12       Fig-13                                          There are some permanent cartilage that do not ossify such as cartilage of trachea and articular cartilage of joints, intramembranous bone formation occur directly without a cartilage template.(4) When preparing double stained specimens, the overlying skin of the specimen must be removed to allow Alcian blue to penetrate and stain the cartilage (13), the skin is difficult to remove from the extremities of fixed specimens so if cartilage formation in digits is of 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.  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 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 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). 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. Dix Davis . D. Dwight and Ulys Roy Gore. Field Mus. Of Natur. Hist. Technique  series No. 4 October 1936 P.1 6. Alcian blue staining of the mouse foetal cartilaginous skeleton. Andras Nagy, Marina Gertsentein, Kristina Vinterstein, Richard Behringer. CSHL Press,     Cold Spring Habor, Ny, USA 2003  K. Leena Pramod et al./Rec Res Sci Tech 3 (2011) 54-58  7. O’rahilly, &Gardner E. The initial appearance of Ossification in staged human embryos. Am J Anat.1972; 134: 291-308 8. Large scale double staining of rat foetal skeletons using Alizarin Red S & Alcian blue. Angela.D Young, Donal E.Phipps, Barry Astroff. Teratology, 6(4); 273—276 9. Simons E.V.,and J.R.Van Horn. 1971. A new procedure for whole mount alcian blue staining of the cartilaginous skeleton of chicken embryos, adapted to the clearing procedure in potassium hydroxide. Acta Morphol. Neerlscand,8:281-292 10. Functional photography, july/Aug 1981, vol 16, no. 4, chapter 22,The visible skeleton. Dr. James Hanken , Dr. Richard Wassersug. 11. Cortes – Delgado.N; Perez-Torres J.R Hoyos J.M. Staining procedure of cartilage and skeleton in adult bats and rodents. Int. J. Morphol, 27(4); 1165-1167 2009 12. Narotsky M.G, Rogers. JM. Examination of axial skeleton of fetal rodents. Developmental biology protocol Vol.1; 139-141  13. Goldstein D. J., R.W. Horobin. Rate factors in staining by alcian blue. Histochem. J.,1974;  6, 157-174. 14. Diagnostic - staining protocol of cartilage and bone, Synnove Helland Wilson Bull, Technique for avian syringes June 1988,100(2); 289-293 15. Taylor, W. R. & G. C. Van Dyke.  1985.  Revised procedures for staining and clearing small fishes and other vertebrates for bone cartilage study.  Cybium9(2):107-119.  

Museum Preservation of Skeleton of Fetus & Small Vertebrates

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Museum Preservation of Skeleton of Fetus & Small Vertebrates

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