Biology, Medicine, & Natural Product Chemistry  ISSN 2089-6514 (paper) 
Volume 8, Number 1, 2019 | Pages: 17-21 | DOI: 10.14421/biomedich.2019.81.17-21 ISSN 2540-9328 (online) 
 

 

 

 

A Simple Method for Clearing and Staining Specimens for 

The Demonstration of Animal Skeleton 

 
Muhammad Ja’far Luthfi1,*, Nyoman Puniawati Soesilo2 

1Department of Biological Education, Faculty of Science & Technology UIN Sunan Kalijaga Yogyakarta, Indonesia 
2Faculty of Biology, Universitas Gadjah Mada Yogyakarta, Indonesia. 

 

Author correspondency*:  
jafarluthfi@yahoo.com 

 

 

Abstract 

 

Teaching skeletal system would be more attractive using real/preserved specimen compared to those using only book. The aim of the 

research was to develop clearing and staining method of animal skeleton specimen using Alizarin Red S-Alcian Blue as teaching aid tool. 

The specimen were eviscerated, fixed, stained, cleared, and keep in glycerine solution. The spesimen will increase effectiveness in 

elementary school science teaching and learning. 

 

Keywords: skeleton; Alizarin Red S; Alcian Blue; elementary school; teaching aid 

 

 

INTRODUCTION 
 

Skeleton is a supporting system for vertebrate bodies. 

Skeleton serves to give shape and support the body, as a 

means for movement, as a protector of internal organs 

and delicate organs (blood vessels and nerve tissue), and 

as a place for muscle attachment. Skeleton is divided 

into axial skeleton and appendicular skeleton. The axial 

skeleton consists of skull and vertebrae (spine), while 

the appendicular skeleton consists of bones in the fore 

limbs and hind limbs (Kardong, 2002). Vertebrates are 

animals that have an endoskeleton and from one of its 

components the name is taken (vertebra/spine). 

The skeletal system is one of the subject taught in 

high school. This material is usually taught by using 

books or pictures. However, according to Prokop et al 

(2007), teaching skeletal systems will be more attractive 

if using original specimens or preserved material 

compared to books. Direct observation plays an 

important role in students' understanding of biological 

phenomena. Students' mental models of a biological 

phenomenon are strongly influenced by the learning 

process (Prokop et al, 2007). 

The National Science Education Standards in the 

USA emphasize that scientific inquiry is the basis of 

science education, where students at all levels should 

have the opportunity to conduct scientific inquiry. 

Experiments are not only provide information about an 

organism's system, but can also develop the ability to 

observe, think critically and experimentally. Students 

who follow the science process will better understand 

science concepts, appreciate how science concepts are 

discovered, and enhance the skills needed to investigate 

nature (Greene & Greene, 2001; Newman et al., 2004). 

Clearing and staining of the skeleton aims to 

demonstrate bone structure and cartilage in animals 

(Erdogan et al., 1995; Inouye, 1976). This method is 

commonly used in the field of teratology to study fetal 

defects. In teratological studies, experimental animals 

used are rat and mouse embryos. Since bone tissue and 

cartilage in embryos and in adult animals are similar 

histologically, and so do the small vertebrates and the 

large ones, theoretically the method for mouse embryos 

can be applied to demonstrate skeletons in adult animals 

or in larger vertebrates (Junquiera & Carniero, 1988; 

Leeson & Leeson, 1981). From its ability to demonstrate 

bone and cartilage structure, this method has the 

potential to be applied for developing skeletal learning 

media. 

The method described in this article is a procedure 

for clearing and staining animal skeleton specimens 

using Alizarin Red S-Alcian Blue as a tool in teaching 

the skeletal system. the procedure can be followed by 

students themselves.  
 
 

MATERIALS AND METHODS 
 

The animals used in this study were goldfish (Cyprinus 

carpio), oriental whip-snake (Ahaetulla prasina), skink 

(Mabouya multifasciata), turtle (Trachemys scripta 

elegans), and flying dragon (Draco volans). The 

equipments used in this study were surgical scissors, 

scalpels, tweezers, and glass jars. The chemical used in 

this study were Alizarin Red S-Alcian Blue, ethanol, 

acetone, aquades, glacial acetic acid, glycerin, and 

KOH. 

The method is a modification of the Inouye (1976) 

originally used for small vertebrates/embryo. Inouye is 

https://doi.org/10.14421/biomedich.2019.81.17-21
mailto:jafarluthfi@yahoo.com


 

 

 

18 Biology, Medicine, & Natural Product Chemistry 8 (1), 2019: 17-21 
 

 

one of the first researchers that successfully use the 

method for bones and cartilage staining. This is a double 

staining method for bone and cartilage of mice for 

teratological studies. Using this method, cartilage 

stained blue, bone stained red, whereas muscles and 

other tissues become transparent. Preparations for 

animal larger than mice and not embryos will need 

modification of a more massive skin and muscle 

removal, additional fixation time, fat removal, and 

extensive clearing and staining due to the thickness and 

robustness of tissue of mature and larger vertebrates.  

Clearing and staining specimens (carp, snake, gecko 

tail, turtle tail, flying dragon) include several stages. The 

first step is exfoliation. After the scales were removed, 

the specimens were fixed in a tube containing 95% 

alcohol for 3 days. Then the skin and muscles in the 

specimen are removed using scissors, tweezers and 

scalpel. Then fixed again in 95% new ethanol for 3 days. 

To remove fat, the fixed specimen was immersed in a 

tube containing acetone for 4 days with the replacement 

of new acetone on the second day. After that the 

specimens were immersed for 5 days in a dye solution at 

37 ° C (1 volume 0.3% Alcian Blue in ethanol 70% + 1 

volume 0.1% Alizarin Red S in 95% ethanol + 1 volume 

glacial acetic acid + 17 volume ethanol 70%). The 

specimens were washed in water and then immersed in a 

solution of 1% KOH for 5 days (to make it transparent) 

and then soaked in succession of a mixture of glycerin 

and 1% KOH at a ratio of 20%: 80%; 50%: 50%; 80%: 

20%. Finally, the specimen were stored in pure glycerin. 
 
 

RESULTS AND DISCUSSION 
 

This research has successfully develop method for 

clearing and staining animal skeleton specimens with 

Alizarin Red S-Alcian Blue (figure 1, figure 2, figure 3, 

figure 4 and figure 5). 

Figure 1 shows a carp skeleton. The preparation 

shows the bones of the skull, vertebrae (backbone), and 

the ray fin of tail of fish that are suitable for live in 

water. Figure 2 is skeleton specimen of oriental whip-

snake. The snake specimens show a special adaptation 

of vertebrate skeletons, that is, very large numbers of 

vertebrae (+ 300 vertebrae) suitable for terrestrial life 

without limbs. Figure 3 shows a skink's tail skeleton. 

The preparation shows the structure of the tail’s 

vertebrae that can autotomized. Figure 4 shows a turtle's 

tail skeleton. Figure 5 shows the skeleton of flying 

dragon. The turtle and flying dragon specimens show the 

structure of the tail vertebra in animals that cannot be 

autotomized.  

Using these specimens, students can directly observe 

the structure of the real specimen, can figure out its 

morphology, know the difference in structure related to 

its function, and gain direct experience about the 

skeleton. Students can more easily understand the way 

of life and movement of snakes and the appearance of 

their elongated morphology by looking at the structure 

of the snake's vertebrae and its enormous number of 

segments compared to fish. Likewise, students will be 

able to understand the relationship between structure and 

function, where an autotomized tail has a fracture plane 

in its vertebrae while animals that cannot autotomy do 

not have a fracture plane in their tail vertebrae. 

According to Eshach & Fried (2005), in science learning 

real specimens have advantages compared to sketches, 

drawings, or books. Real specimens will attract more 

students. In addition, original specimens have the 

advantage of 3-dimensional detail that is not possessed 

by sketches or photographs. While skeleton props often 

do not have the details according to the original 

preparation.  

 

 
 

Figure 1. Structure of a carp skeleton. Visible skull bones, spine, and rays 

of fish tails. (a) Tail rays; (b) Vertebra. 

 

 

 

 
 

Figure 2. (A) Complete view of snake skeleton. (B) Higher 

Magnification. Skeletal structure of an oriental whip-snake. There are 

skull bones, vertebrae with very large amounts of ribs. Note the spine with 

ribs. (a. Ribs; b. Vertebra). 

b

a

b

a



 

 

 

 Luthfi & Soesilo – A Simple Method for Clearing and Staining Specimens for … 19 
 

 

  
 

Figure 3. (A) Skink's tail skeleton structure. Visible vertebrae/tail bones with their processes. Note that there are fracture plane in the bone where the tail 

breaks off during an autotomy. (B) Skeleton of regenerated skink tail. After autotomy, skink will develep regenerated tail which the cartilaginous tube 

(stained blue) supporting tail instead of bony vertebrae. (a. Neural spine; b. Centrum; c. Fracture plane; d. Intervertebral pad; e. Chevron bone; f. Vertebra; 

g. Cartilaginous tube).  

 

 

 

 
 

Figure 4. High magnification of the turtle tail skeleton structure. The tail 

consists of a series of vertebrae with their processes. The turtle's tail 

cannot autotomy and therefore the turtle's tail vertebrae do not have a 

fracture plane. (a. Centrum; b. Chevron bone). 

 

 

 

 

 
 

Figure 5. High magnification of the flying dragon tail skeleton structure. 

The tail consists of a series of vertebrae with their processes. The flying 

dragon tail cannot autotomy and therefore do not have a fracture plane. 

(A). Dorsal view. (B). Ventral view. (C). Lateral view. (a. Neural spin; b. 

Tranverse process; c. Centrum; d. Chevron bone).  

 

The results show clearly visible bone structure in 

goldfish, oriental whip-snake, gecko tails, flying dragon 

tail and turtle tail. The bones stained red while the 

cartilage stained blue. Muscle and connective tissue 

become transparent/clear by the clearing process using 

KOH. In these preparations the details of the skeleton 

can also be observed such as the processus of the 

vertebrae, intervertebral discs, ribs, fracture plane, and 

other details. 

Figure 1 shows a fish's skeleton. Students can 

observe the presence of skull bones in fish, vertebrae, 

and fin rays of fish. From these specimens, it is known 

that the fin rays of the fish are composed of bone 

material. From the snake preparation (figure 2) we can 

observe the structure of the snake vertebrae that has ribs 

on its body vertebrae. Only in the tail, the snake 

vertebrae do not have ribs. The ribs serve as muscles 

attachment and help the movement of snakes. The skink 

tail (figure 3), turtle tail (figure 4) and flying lizard tail 

(figure 5) provide structures that are ideal for studying 

skeleton anatomy, and to some extend student can learn 

the relationship between form and function. Skink's tail 

can autotomized its tail, whereas the tail of a turtle and 

flying lizard cannot autotomy. This functional difference 

is also reflected by differences in structure, namely the 

existence of fracture plane in the skink tail vertebrae. It 

is on these fracture planes that the tail can break. This 

autotomy ability is a form of gecko's self-defense to 

avoid predator. Turtles and flying lizard cannot 

autotomized its tail so that it can be understood if there 

are no fracture plain in the vertebrae. Adapting its way 

of life, the tail of the flying lizard is needed to help the 

movement on the tree and when it gliding. Therefore, 

breaking the tail would certainly be less profitable. The 

same is true for turtles. Turtles have very hard carapace 

and plastrons. If there is a threat or an enemy, the turtle 

g

f

e

b

a

d

c

b

a

d

cba

b



 

 

 

20 Biology, Medicine, & Natural Product Chemistry 8 (1), 2019: 17-21 
 

 

will insert its head and motion apparatus into the 

carapace and plastron. 

Learning in this way will be more interesting for 

students. This bone specimen makes it easier for 

students to understand skeletal structure. Understanding 

the anatomical structure will facilitate the understanding 

of its function. Students will be able to observe the 

components of the skeleton, the skeleton differences in 

its way of life, the skeleton differences between animal 

classes, and the relationship between structure and 

function (a given structure reflects certain function). In 

addition, using real specimens will be more interesting 

for students related to their 3-dimensional shapes, 

attractive colors, and direct experience of seeing original 

specimens from a visual, tactile and motoric perspective.  

Appleton (2008) stated that often school teachers are 

hesitant in teaching science. The main reason is the 

limited knowledge of teachers about science content. 

This paper provides a teaching of animal skeleton by 

directly involving teachers and students in the science 

process so that they can see directly the science process. 

Thus the confidence of teachers and students will arise 

to learn science. Matthews et al (1997) stated that 

students have a high curiosity about the world around 

them, and nature offers a variety of materials in which 

the teacher becomes a mentor to help students to satisfy 

their curiosity by studying the natural surroundings. 

This study used local animals that are widely found 

around us, namely, goldfish/carp (Cyprinus carpio), 

oriental whip-snake (Ahaetulla prasina), gecko (Gecko 

gecko), turtle (Trachemys scripta elegans), and flying 

dragon. This certainly makes it easier for teachers to 

develop science learning media in accordance with 

school availability and abilities. This is in accordance 

with the opinion of Matthews et al (1997) which stated 

that nature provides the appropriate material to satisfy 

students' curiosity that is so high towards the natural 

surroundings. 

According to Eshach & Fried (2005), the superiority 

of the real specimens for learning as mentioned above is 

very suitable for children's learning. Children (including 

elementary school students) have a high curiosity. This 

character is very suitable for science considering that 

one of the factors driving science is curiosity. Children 

really like to observe and think about the environment. 

Exposing scientific processes to young persons will 

develop positive attitudes towards science (Eshach & 

Fried, 2005; Appleton, 2008). Greene et al (1993) stated 

that real specimens are very important in many aspects 

of cognitive learning in the classroom. 

In this study the most important aspect in the success 

of staining is skin and muscle removal, and the stages of 

fixation. The skin and muscles must be removed as 

much as possible so that only a little muscle remains for 

the attachment of each bones. But it should be noted that 

skin and muscle removal should not cut the 

bone/skeleton itself. The vertebrae have many processes. 

Often during the removal of skin and muscles, the 

process of the vertebrae is unintentionally cut off. This 

will certainly change the vertebral morphology caused 

by the artifact, as a result of error in cutting. 

Fixation and fat removal were done longer than the 

Inouye method, because the structure of the skin and 

muscles of the mature animals are thicker and stronger 

than the embryo so that the fixative solution takes longer 

to penetrate the tissue. Different specimens require 

different time periods for fixation, fat removal, clearing 

and staining. This depends on the species, maturity 

(embryo or adult), body size, and organs/body parts 

made for preparations. In general, larger and more 

mature animals require a longer period of fixation, fat 

removal, clearing and staining. The length of time for 

fixation, fat removal, clearing and staining in this study 

can be used as a guide in staining for other animals. 

This research has succeeded in developing a skeleton 

(bone and cartilage) staining method for learning 

skeleton systems for high schools. Learning in this way 

will involve the teacher and students in the science 

process. In addition, the use of real specimens will make 

it easy to understand the skeleton stucture and more 

attractive to students. The process and materials needed 

in this study are very simple, and can be applied to other 

local animals that are available according to the 

availability in the environment around the school. 
 
 

CONCLUSIONS 
 

The study of organisms including the skeletal structure 

is an important part of the basic education science 

curriculum. The use of pictures, sketches, visual aids, 

and books to explain the skeletal structure is a common 

method used in the teaching of skeletal systems. 

However, this method often causes less student interest. 

The skeleton system is also more difficult to understand 

with the use of instructional media. The author provides 

alternative for high school teachers in teaching the 

skeletal system. Making skeletal preparations by 

involving students is not commonly carry out in the 

learning of primary school framework systems, but 

teachers can combine them with the existing curriculum 

to expand the scope of basic education. 
 
 

ACKNOWLEDMENTS 
 

The authors would like to thank the assistants of Animal 

Anatomy Lab Work at the Faculty of Science and 

Technology of UIN Sunan Kalijaga, members of the 

Apprenticeship Program at the Faculty of Science and 

Technology of UIN Sunan Kalijaga, and members of the 

Center for Integrative Zoology of UIN Sunan Kalijaga 

for their role to make this research possible. The authors 

also would like to thank to Mr. Riyanto (staff of the 

Faculty of Science of Technology) for his assistance in 

pictures processing. 
 



 

 

 

 Luthfi & Soesilo – A Simple Method for Clearing and Staining Specimens for … 21 
 

 

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