Plastination of Some Cow and Ram Organs in Ghana for Use as Teaching Aids

Headnote

Abstract: The plastination procedure was adapted using locally available materials in Ghana to prepare plastinated specimens of the heart and kidneys of a Cow, the lungs and attached trachea of a Ram, and the penis and attached testicles of a Ram for use as teaching aids. The extent of shrinkage varied among the various organs and among the various stages of plastination. For the individual specimens the extents of shrinkages at the beginning and at the end of each plastination stage were not significantly different (P≤0.05) but significantly different (P≤0.05) among the various stages of plastination. The extents of shrinkage among the various organs were not significantly different. Shrinkage was highest at dehydration and the least at fixation. With the exception of the Ram's lung which discoloured and lost its natural shape the plastinated specimens maintained their shapes and are satisfactory for use as teaching aids. Plastination could easily be adapted for preparing teaching aids for science classes at all levels of education in Ghana.

Keywords: Teaching-aids, Plastination, Shrinkage, Colour, Texture.

(ProQuest: ... denotes formulae omitted.)

1. Introduction

The uses of biological specimen are important as teaching aids for lessons in anatomy and physiology and other related subjects. The specimen may be fresh or preserved, however fresh specimen are preferred because they illustrating the gross anatomy of the specimens more vividly than the preserved specimen. However, the fresh specimens soon lose their form through decay, and the only way to prolong their use is to preserve them. Traditional methods of preservation include drying, immersion in chemical preservatives or perfusion of blood with chemical preservatives. However, dried specimens lose their colour, and become darker and easily crumble. The prolonged use of chemical preservatives may prove toxic. Long exposure to formaldehyde can cause mild neurological symptoms, including headaches and dizziness, and genetic damage (Mirabelli et al, 2011).

Zenker's fixative is a rapid nuclear fixative used as a mordant in some staining procedures and is used to prepare animal or vegetable tissues for microscopy. The composition is 5 parts mercuric chloride, 2.5 parts potassium dichromate, 1 part sodium sulfate, and 100 parts water. High concentrations of mercuric chloride have been shown to have adverse effects on the motility of spermatozoa in rabbit semen (Slivkova et al, 2010). Osmium tetroxide is a secondary fixative for preparing samples for electron microscopy, but it is toxic to the lungs and the nervous system.

Plastination is a patented and novel method of preservation of biological specimen developed by Dr. von Hagens in 1977 at the department of Anatomy of Heidelberg University in Germany. It is especially good for preserving gross biological specimen and involves the replacement of the water and lipids in the specimen with plastic-like polymers. The final products are of superior quality than those preserved by other methods, retain most qualities of the original sample (Weiglein, 2005) and are dry, odorless, non toxic and can be handled with bare hands (Lee et al, 2005). Plastination is used for the preservation of perishable biological specimens, especially for soft, putrifiable organs with high water content like ranging from a full human body to a small piece of an animal organ like the brain, heart, liver, muscle, and thin organ sections and body slices and also plants and insects (Cook, 2005) and the final products are called Plastinates (Priya et al, 2007).

The plastination procedure consists of four main steps of plastination which follow specimen dissection and preparation (Weiglein, 2005). In the first step which is fixation (Henry et al, 1997) the specimen is immersed in a chemical preservative usually in a formaldehyde solution to halt decomposition. Dehydration then follows by placing the specimen in a bath of acetone under freezing conditions. The acetone draws out all the water and replaces it inside the cells (Bickley et al, 1990). In impregnation, the specimen is submerged in a bath of liquid polymer usually silicone rubber, polyester or epoxy resin, under vacuum, causing to acetone to boil at a low temperature and vapourise out of the cells. Simultaneously, the plastic liquid polymer diffuses into the cells and occupies the spaces leftby the acetone (Bickley et al, 1990). In the final step the specimen is cured with gas, heat, or ultraviolet light and this hardens the plastic liquid polymer inside the cells making the whole specimen to become hard and plastic (Henry et al, 1997).

It is reported that over 250 institutes for Human Anatomy, Clinical Pathology, Biology and Zoology worldwide now use plastination for preserving biological specimen for use as teaching aids (Sora, 2005). At the University of Michigan Medical School, plastinated specimens have become an essential part of medical, undergraduate and dental anatomy education (Raoof et al, 2005). However, there are no known reports of the preservation of biological specimen by plastination, or the use of plastinates for teaching in Ghana.

In this study, the plastination procedure was adapted using locally available materials in Ghana to prepare plastinated specimens of the heart and kidneys of a Cow, the lungs and attached trachea of a Ram, and the penis and attached testicles of a Ram.

2. Materials and Methods

Specimens

The heart and kidneys of a Cow, the lungs and attached trachea of a Ram, and the penis and attached testicles of a Ram were used for the study. The organs were obtained immediately after slaughter of the animals at the Accra abattoir and after they had been certified safe for consumption by a Veterinary Officer of the Accra Metropolitan Authority. The organs were immediately placed in crushed ice in an ice chest and brought to the laboratory.

Fixation, Washing Out and Dehydration

All specimens were washed with tap water to remove blood and dirt.

A 10% formaldehyde solution was poured into the lungs to its capacity through a funnel placed at the mouth of the trachea. The cavities of the heart and trachea were stuffed with small balls of cotton wool soaked in 10% formaldehyde solution (Fig 1) and together with the other specimens totally immersing in 10% formaldehyde solution for 16 days at room temperature. The heart and lungs were emptied of the formaldehyde solution and then immersed with all the other specimens in a bowl of water placed under gently running tap for 24 hours to wash out the fixative. The specimens were dehydrated in 50% acetone solution followed by 80% acetone solution each for 24 hours at -4°C in a refrigerator, and then finished offin 100% acetone for 5 days at -4°C in a refrigerator.

Pre - Impregnation with silicon polymer

The specimens were pre-impregnated at -4°C with silicon. This was done by immersing them in a liquid silicon polymer bath consisting of 40% (w/v) Silicone (ST 368 brand) in Xylene in a Mikachi refrigerator for 3 days to allow excess acetone to escape and the specimens to equilibrate naturally with the silicon solution. Weights placed on the specimens kept them totally submerged.

Intermittent Forced Impregnation with silicon polymer

Intermittent Forced Impregnation was done at -4°C for 13 days in a vacuum chamber that was created by attaching one end of a flexible rubber tubing to a sealed container containing the specimens immersed in the silicon polymer solution (Fig 2). The other end of the tubing was attached to a Rotary Vane Vacuum Pump (Type RE 5) which was switched on for 12 hours and then offfor 12 hours, for 13 days. The vacuum created by the suction caused the acetone to vaporize out of the cells and created spaces for the polymer to diffuse into the specimens.

Post-Impregnation

The vacuum pump was switched offand the desiccator disconnected from the vacuum pump. The specimens were kept submerged in the silicon solution in the sealed container placed in the refrigerator maintained at -4°C for 4 days.

Curing and Hardening

A solution of Xylene Hardener (Sikkens 1.2.3 brand) was used to coat the internal and external surfaces of the impregnated specimens which were then placed in an open container and exposed to UV light in a sterilisation chamber at room temperature for several days. The completed specimens were stored in a sealed Plexiglas frame to protect them from dust.

Measurements for Levels of Shrinkage

The following were selected for evaluating the levels of shrinkages at the various stages of plastination: the Cow Heart, Cow Kidney, Length of Ram's Trachea, Internal Diameter of Ram's Trachea, and External Diameter of Ram's Trachea.

The distance Fre1 (mm) between two clearly identifiable spots at one area on the surface of each fresh specimen was measured. This was repeated at two other separate areas on the surface of the specimen to obtain Fre2 and Fre3.

The mean distance Fre was then calculated as

... (1)

There are five stages involved in plastination: Fresh sample, Fixation, Dehydration, Impregnation, and Curing.

At each stage of plastination the distances B1, B2 and B3 respectively were taken between the same sets of spots at the beginning of the stage and the mean distance B calculated as

... (2)

At the end of the stage measurements E1, E2 and E3 respectively were taken again between the same sets of spots and the mean distance E calculated as

... (3)

The shrinkage S was calculated as

S = B - E (4)

The fractional shrinkage of the fresh sample Sf was calculated as

Sf = S / Fre (5)

Where Fre = B for the fresh sample

The percentage shrinkage Sp was calculated as

Sp = (S / Fre) x 100 (6)

The results are indicated in tables 1 - 5.

The mean percent shrinkage for each stage and the total percent shrinkage for each specimen (Table 6), and the percent cumulative shrinkage along the process (Table 7) were calculated.

Statistical Analysis

Statistical analysis was done for significant differences (P≤0.05) between the beginning and end stage measurements, and for significant differences (P≤0.05) in measurements among the various stages and among the various specimens.

3. Results and Discussions

The extent of shrinkage varied among the various organs and among the various stages of plastination (Tables 1 - 5). The extent of shrinkage ranged between 0.93% - 4.19% for the bovine heart, 0.43% - 2.60% for the bovine kidneys, 0.005 - 1.63% for the length of Ram trachea, 0.00% - 5.56% for the Internal diameter of the trachea, and 0.00% - 4.76% for the External diameter of the trachea (Table 6). For all the specimens' shrinkage occurred the most at dehydration and the least occurred at fixation. The mean percent shrinkage at dehydration was 3.75% and 0.36% at fixation (Table 6).

The specimen which shrunk the most was the bovine heart (6.98%) and the length of the trachea experienced the least reduction of 3.27% (Table 6).

From fixation, through dehydration, impregnation to curing the extent of accumulated shrinkage progressively increased for the trachea length, kidney and heart (Fig 10). The internal diameter and external diameter of the trachea progressively shrunk during fixation to dehydration, but experienced no significant changes during impregnation and curing (Fig 10). For the individual specimens the extents of shrinkages at the beginning and at the end of each plastination stage were not significantly different (P≤0.05) but significantly different (P≤0.05) among the various stages of plastination (Tables 8 - 12).

The extents of shrinkage among the various organs were not significantly different (P≤0.05) (Table 13). The different specimens are made up of different tissue types and arrangements. The trachea is made of incomplete cartilage rings, the heart consists of specialised cardiac muscles made up of branching striated cells, the lung tissue consists of a mass of spongy tissue containing an anastomosing network of capillaries, and the kidneys contain millions of microtubules (Mader, 1998). However, the responses of the various specimens to the treatments at the various stages of plastination were not significantly different. This means that generally the various specimens experienced the same levels of treatment during the whole plastination process.

The stability of proteins may be enhanced by reacting with other substances. Nazarudin, (2011) showed that when a chitosan which is a protein is blended with agar which is a galactose polymer the heat stability of the blend is higher than that of either the chitosan or the agar. In this study the specimens were fixed with formaldehyde which dissolves rapidly in water to form Methylene hydrate, HO-CH2-OH. Methylene hydrate molecules react with one another to form polymers. Initial binding of formaldehyde to protein is largely completed in 24 hours but the formation of Methylene bridges proceeds much more slowly (Kiernan, 2000) producing an insoluble high molecular weight complex (National Diagnostics, 2010).

The plastination of the Ram lungs was not satisfactory. The lungs maintained their shape at full vital capacity during fixation, dehydration and impregnation. This was because the solutions of formaldehyde, acetone, and silicon polymer respectively were poured into the lungs through the trachea and filled the cavities to capacity thereby maintaining the shape as at full vital capacity during fixation, dehydration and impregnation respectively. However, at the end of impregnation the silicone polymer solution was poured out of the lungs. The curing of the lungs therefore occurred in the absence of a liquid medium within the cavities. The lungs were therefore not distended during curing and rather shrunk rapidly and extensively. The internal structure of the lungs consists of millions of alveoli air sacs which form a spongy, elastic, porous and light texture. This structure is maintained by the presence of air in the alveoli. Therefore the absence of an alternative medium in the alveoli during the curing stage to maintain the shape of the lungs resulted in the deformation of the lungs at the end of curing. The lungs were also discoloured from pink to brown, and had lost their natural shape (Fig 3). The plastinated lungs lost the sponginess of the fresh specimen and became firm and solid. Curing changed the liquid silicon polymer into a solid. If the cure period is longer, the solid silicone molecules would be longer and the specimen would be more flexible (Henry, 2005). The plastinated Ram lungs obtained in this study are therefore not good for use as a teaching aid.

According to (Henry, 2005; Raoof, 2005; Slivkova et al, 2010; Boyes and Kippers, 2005), hollow organs must be dilated during fixation as well as during dehydration and curing to produce organs such as the lungs, stomach and intestines with realistic sizes and shapes. During dehydration, the acetone drew out all the water and replaced it inside the cells (Bickley et al, 1990). The shrinkage of specimen by dehydration by acetone can be minimised if the procedure is done at -20°C (Raoof et al, 2005; Bickley et al, 1990). A better method of curing the lungs would therefore have to be explored in subsequent studies.

The physical characteristics (shape and colour) of the other plastinated specimens (bovine heart and kidney, and Ram trachea, and Ram penis with attached testicles) obtained at the end of the experiment makes them suitable for use as teaching aids. The cow kidneys turned brown (Fig 3) while the plastinated rams penis and attached testicles are white (Fig 7) and very dry and solid. The scrotal sacs have a papery texture with the testicles hanging loosely in them.

The cotton stuffed into the heart was able to keep the four major blood vessels that transport blood to and from the chambers of the heart open. The shape of the heart was also maintained. Further dissection of the specimens is possible after plastination and this would enhance the features displayed (Boyes et al, 2005).

The colours of the final products were markedly different from those of the fresh specimens. All the plastinated specimens lost the pink look of the fresh specimens and this was due to the loss of blood from the specimens through diffusion into the surrounding medium during fixation, and dehydration. It is important to keep the fixation period as short as possible in order to preserve as much as possible the natural colour of the specimen (Henry et al, 1997; Bickley et al, 1990). For this study fixation was for 16 days at room temperature.

This study statistically confirms the report by (Ilieski et al, 2005) that many researches had deduced that the shrinkage of specimens occurs during dehydration and impregnation. In this project dehydration was done at -4°C but according to (Weiglein, 2005) complete dehydration must be done at -20°C to avoid shrinkage. Forced impregnation is the most important stage of the entire process and must take place for as long as possible. This can be achieved if the silicone polymer has a low viscosity (Bickley et al, 1990). Fast impregnation causes shrinkage mainly in thick specimens because the polymer cannot be forced into the cells as fast as the acetone leaves them.

The main equipments used for the plastination are plastic containers for fixating, dehydrating, and impregnating the specimens in; a deep freezer for providing a low temperature for the fixative, dehydrating, and impregnating agents; a UV bulb for curing the impregnated specimen; and a Rotary Vane Vacuum Pump (Type RE 5) and flexible rubber tubing. All the chemicals used in this study were obtained locally in Accra, Ghana.

Large amounts of acetone were used in this project for dehydration. Dealing with the used acetone solution was a problem. In order to protect the environment and reduce operation cost, it is recommended that the acetone be recovered for use in the laboratories.

4. Conclusion

The plastinated specimens with the exception of the lungs are satisfactory for use as teaching aids. The study shows that locally available materials and appropriate technology in Ghana can be used to prepare plastinated specimens for use as teaching aids. The results are encouraging especially this being the first known reported experiments on plastination in Ghana. The plastinates would be used as teaching aids at the Department of Science Laboratory Technology of Accra Polytechnic, Ghana.

Acknowledgments

This study is being supported by a research budgetary allocation from Accra Polytechnic, Ghana to the Department of Science Laboratory Technology of Accra Polytechnic.

References

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