Plastic Embedding of Zoological Specimens
The value of plastic embedded specimens as a teaching aid is now well recognised. It was to take advantage of these benefits, especially as a replacement for wet-mount bulky specimens, that a programme of embedding was initiated in the Zoology Department, Victoria University. From instruction leaflets, etc., the technique appeared to be a simple one, with little or no problems to trap the unwary. This we found to be true to a limited extent for the range of teaching material required in the department.
The teaching material we wished to embed covered a wide range both in surface and bulk texture and in size. The material varied from hard, barely porous shells and teeth to the open meshwork of the Porifera, through bone and cartilage to chitin-covered arthropods or coelenterates, and to delicate jellyfish or the early stages of the chick blastodisc. The largest specimen we have so far embedded is an eviscerated mammalian embryo 15 × 5 × 3.5 cm in which the skeleton had been stained with Alizarin Red S to distinguish the ossified tissue. The unpolished plastic block for this specimen measured 23 × 5 × 10 cm. The smallest individual specimens embedded were unstained copepods, 3-4 mm in length.
In the course of overcoming our difficulties with the embedding of such material, we also tried and found successful other procedures for sectioning the embedded material, and for block shaping and polishing. Comments on these procedures and on the embedding techniques we finally adopted we felt might be of interest and assistance to other workers contemplating plastic embedding of a similar range of zoological material. The quantities of material and the timing given for the embedding processes apply to polyester resin Au 8018C, styrine monomer and catalyst M.E.P.K., as manufactured by A. C. Hatrick (N.Z.) Ltd., Tawa, Wellington.
Figs. 1 & 2 Formica faced L-pieces placed to form a mould.
Fig. 3 Sectional view of mould to show the approximate degree of "tackiness of the resin surface prior to the pouring of the next layer.
Fig. 4 Metal-sided, 4 in one mould showing also the glass cover plate.
Flexibility of usage from a single "box" mould for large projects (Figure 4) is obtained by the provision of metal inserts, giving a one, two, three, or four-in-one box. Ease of removal of the blocks from this type of mould comes from the ability to open out the sides. The glass base and top plate give a surface to the hardened block that needs very little polishing. Virtually no presurfacing of the glass or metal with a releaser compound such as silicone wax is necessary as the plastic shrinks away from the mould during hardening.
Basically, the whole process of embedding can be summarized in four words: dehydrate, clear, embed the specimen and polish the plastic block. But before proceeding with a project, if you wish to admire your handiwork as a plastic block which has a high surface gloss, is almost crystal clear, and without bubbles, it is necessary to possess considerable patience and be aware of some of the problems that may be encountered in carrying out the four basic techniques noted above.
A. Dry material
It is very desirable to know something of the past history of the material to be embedded. This is particularly the case with specimens that have been stored dry such as insects, crabs, spiders, corals, teeth, etc., but may have been subjected to fixation and/or temporary storage in liquid. If the past history of the specimen is not known, dry material is best placed in acetone under vacuum until all the air is removed, and the specimen thoroughly penetrated. If the specimen is large, this may take upwards of an hour utilizing vacuum pressure at 20-minute intervals. The specimen can then be redried or placed in acetone for storage prior to dipping it in the styrine monomer at the next stage in the embedding procedure (see also p. 4 for arthropod material).
B. Wet preserved specimens
The material should be thoroughly washed free of preservative and then dehydrated through 50% to 100% acetone. The length of time in which the specimen remains in the two grades of acetone depends, as with alcoholic dehydration, on the size and density of the specimen. In general, however, specimens of a similar size need less time in an acetone dehydration series than in an alcohol series. Acetone has another advantage over alcohol for dehydration in that it retains the colour of a wider range of specimens, particularly arthropods, than does alcohol. Furthermore, it does not matter for plastic embedding if the specimen is considerably hardened in texture by the acetone. In fact it is advantageous with soft bodied material as such material is then better able to withstand the pressure and heat of the setting resin without distortion.
This two-stage dehydration series proved quite satisfactory for all the material we have so far embedded, with two exceptions. These exceptions are echinoderms, and material that has been macerated in potassium hydroxide. With echinoderm material, a test specimen should first be tried in acetone, as acetone decolourizes and to some extent decalcifies many echinoderms. We have found it better to fix in formalin, wash in water, and then oven dry the specimen. Specimens that have had the tissues page 4macerated in potassium hydroxide need a more gradual dehydration than is afforded by the two-stage series. An 80% acetone bath should also be included in the series. Moreover, it will already have been appreciated that there will also be considerable shrinkage of this material. However, we found that both the monomer and the un-catalyzed resin restore in large measure the lost tonicity of the tissues.
It is also very advisable during dehydration to remove any trapped air in the specimen. This procedure is necessary particularly for material with an open mesh-work support, such as that found in the Porifera.
Any further preparation of the material prior to embedding now depends on the morphological features required for display. For example, with arthropods it is usually the exoskeletal features, but with soft-bodied animals it is more often the internal morphology. With the completion of dehydration in acetone, arthropods in which the exoskeletal features are required need only be dipped for a few minutes in the styrine monomer and they are ready for embedding in the catalyzed resin. Soft-bodied animals and any arthropods required for observation of internal morphology should be cleared to at least semi-transparency.
Clearing the Specimen
The general procedure we adopted was to clear the tissues to semi-transparency by placing the specimen in the styrine monomer. (The catalyzed resin completes the clearing process). The specimens may again need to be placed under vacuum, and should be allowed not only to sink to the bottom of the container but to remain there for some hours longer. Overnight is satisfactory for small specimens of dimensions 7 × 5 × 3 cm. This ensures a thorough penetration of the monomer into the tissues.
We use, however, a slightly different technique for specimens that have been macerated in potassium hydroxide. With this material we have a further series of clearing baths. The first bath is a 1:1 mixture of monomer and polymer. The second a 1:3 mixture and the final bath a 1:6 monomer/polymer mixture. Again the specimens should remain in each grade of the series for two or three days at least after they have sunk to the bottom of the container. The mammalian embryo (noted above on page1) remained in the final stage of the clearing series for 14 days after it had sunk to the bottom. This technique not only assists materially in giving and retaining tonicity in the specimen, but it eliminates air bubbles that may remain even after vacuum treatment, beneath the skin and in the oral and nasal cavities.
Nonetheless, we have not had 100% success in eliminating air bubbles in the hardened block with this type of material (Fig. 12). This is particularly the case with young embryos 2 to 5 cm in length that were not eviscerated prior to maceration and in which the newly formed bone is very porous. Our main problem has been the prevention of very tiny air bubbles forming in the marrow cavities and lungs. This may happen even from one to three days after the block has set sufficiently hard to be removed from the mould, but not hard enough for polishing. The after-clearing method we have found most successful is given on p. 6 under the heading "Insertion of the specimen in the catalyzed resin".
Embedding in Plastic
(a) Preparation of the mould: We have found that moulds or parts of a mould made of metal formica or glass, need only be greased with a very thin film of releaser compound such as silicone wax. During the exothermic hardening process, the plastic contracts sufficiently from the walls of the mould for the completed block either to fall out, or be released by gentle leverage with a flat thin blade between the mould wall and the block. But we do use a thick layer of silicone wax or plasticine sealing page 5compound at the junctions of the L-piece moulds or the internal partitions and between the base of the sides of the mould and the base plate to give a tight seal to prevent loss of the plastic (Figures 1 and 2).
(b) Preparation of the resin: After several months of trial we have found the following procedure to be satisfactory. It has produced clear, hard blocks of plastic. We prewarm to 50 °C in the oven the glass container for receiving the catalyst. (The hardening process once started is irreversible, so only sufficient resin should be mixed with the catalyst to give the desired layer depth. It is unwise to exceed a depth of half an inch for any one layer). Then add the correct amount of resin to the correct amount of catalyst in the container. With resin AU 8018 C it is 0.5 ml of catalyst for every 50 ml of resin. Stir gently with a glass rod until the resin and catalyst are thoroughly mixed. When the catalyst is stirred in, a distinct colour change takes place from the pale blue of the uncatalyzed resin to pale green. The recommended volume given in the instructions supplied with the catalyst is in general too great for biological material. With the recommended volume of hardener, the heat generated during the hardening process is such that the resin tends to "boil" and the fully matured block has a yellowish hue. This colour however may also be in part the result of substances released from the specimen as the resin heats up.
We next put the mixture of resin and catalyst, sufficient to give a layer ¼" in depth, into the oven at 50°C for 3-5 minutes. [The reason for this time range is that different batches of resin vary in the time taken to the commencement of setting. It is advisable to test run a small amount of mix with each new batch of resin. The batch number is marked on the container supplied by the manufacturer.] Now gently stir the warmed plastic with a glass rod. After stirring it is ready to pour into the mould. When pouring is completed, put the mould back into the oven for 10 to 15 minutes. No damage occurs to the caulking plasticine in this time. Return of the newly poured plastic to the oven for a short time brings stray bubbles to the surface to burst, hastens the hardening process and ensures a hard clear outer layer for the block. Further hardening should be continued out of the oven until only the surface is tacky (Figure 3). Cover the mould to prevent dust settling on the hardening plastic. Should the hardening process be inadvertently carried further and the surface become quite hard, we have found that the next molten layer keys on quite well if the surface is covered with a shallow layer of acetone for 2 to 3 minutes. This prepares the surface for a better take of the next resin layer. The junction between the layers is then only visible in side view.
The block is now ready for another pouring of a ¼ to ½ inch layer of catalyzed resin, prepared as above. When the layer has been poured, any air bubbles can be surfaced with a mounted needle and moved to the edges and burst.
(c) Insertion of the specimen in the catalyzed resin: Take the specimen from the preparatory fluid in which it is held and place it on the surface of the newly poured layer. Let it sink into the layer under its own weight. As noted above, the mould can then be returned to the oven for up to 15 minutes. Remove any air bubbles that still remain. Then allow this layer to harden at air temperature, either till hardened right through if the specimen is adequately covered by at least ¼ inch of molten plastic or, if it is not completely covered, until the surface is tacky. If the specimen is not completely covered, continue layering with the resin.
With deep specimens that cannot be covered with one pour of the catalyzed resin, the specimen should, if possible, be positioned in the mould so that apertures such as the mouth and nostrils are completely covered when the specimen is first placed in the resin or alternately completely covered, during the pouring of a single layer. page 6Otherwise these apertures may allow a major ingress of air into the specimen during the hardening of the plastic. Internal air bubbles resulting from leaving these apertures exposed do not always become apparent until the block is too far advanced in the hardening process for them to be successfully removed.
We use a different method for specimens that have been stained to show the skeleton. We have attained our greatest success with this material by lengthening out the time taken for the catalyzed resin to set to a stiff gel. We have tried two methods. In the first, we proceed as above to the pouring of the layer in which the specimen is to be placed. Put the specimen in this layer and return the mould to the oven for not more than 3 to 7 minutes. The lower time range should be used for small specimens. Cover the mould and allow the plastic to harden at room temperature. Follow a similar procedure for any other layers that need to be poured. In the second method we proceed as above only as far as the lower protective layer. The succeeding layer to take the specimen is prepared without heating the vessel to contain the catalyzed resin, and the resin and catalyst are mixed and poured at room temperature. After inserting the specimen in this layer, the covered mould is placed in the refrigerator for not more than 30 minutes, and the layer further hardened at room temperature. The procedure for any succeeding layers is the same. All in all, the first method has so far given the best results. The plastic of the block is clear, not tinted, but a few tiny bubbles may appear in the marrow cavities. In the second method the hardened block is usually a pale straw colour, but tiny bubbles occur less frequently.
Trimming and Polishing the Hardened Plastic Block
Block trimming is virtually unnecessary when moulds such as those described above are used. But should trimming be required, a hacksaw with a fine toothed blade can be used.
The procedure generally advocated for producing a high gloss surface to the block is to hand polish first on a graded series of emery paper, then jeweller's rouge and finally a metal polish such as Brasso. We found hand polishing so time consuming even for a small block (2 × 3 × 4 cm) that the prospect of using this method on blocked material of dogfish skeleton sufficient for a class of 150 students became very unattractive both from the point of view of economy and the sheer physical effort involved.
The answer to both problems came with the installation of a Linisher belt sanding machine. Various small modifications to this sander (Figure 5) allow the polishing process to go on virtually unattended. Also the addition of a vacuum suction tube to the apparatus permits the removal of troublesome plastic fluff and dust from the atmosphere. We used a coarse belt (No. 50), medium belts (No. 60 and 80) and a fine belt (No. 100). The blocks were then polished on plywood discs (Figure 5) to which hat felt had been glued. We soaked the felt on one disc with Brasso and the second with Silvo. The first polish was with the Brasso followed by the Silvo. A vigorous rub on a Dacron surface gives a high gloss finish, or the surface can be spray-coated with plastic.
Fig. 6 Chick embryo to show one method of storing embedded specimens in a plastic bag.
Fig. 7 Weta—Hemideina thoracia (Orthoptera).
Fig. 8 First embedded specimen of an alizarin preparation to show the skeleton of the flatfish Rhombosolea retiaria (alizarin preparation by J. Manikiam, Zoology Department, V.U.W.).
Fig. 9 Peripatoides novae-zealandiae (Onycophora).
Fig. 10 Cicada—Melampsalta muta (Homoptera).
Fig. 11 Hydrocoral.
Fig. 12 Enlarged view of Rhombosolea retiaria. ab = air bubbles.
Secondly, we also found that hardened plastic blocks turned very well on the metal lathe. This allows sections to be made of the specimen at various levels and through a variety of planes. We have not as yet experienced any difficulty in keying on an entirely new surface to the block or filling the cavities made at various levels through the specimen. Cavities, however, need careful cleaning out with acetone, and acetone should be allowed to remain in the cavities for 2-3 hours before the new plastic is poured into them. The junction lines between the original hardened block and the newly made pour can usually be detected, but they do not as a rule impair viewing of the specimen under the binocular microscope.
Another method of making sections is to grind down the block on the sander using the coarse belt. This is a particularly useful technique for sections of hard material such as teeth, bone, or corals. The block is polished in the usual way and either resurfaced with a very thin layer of plastic, or the block is surface sprayed with plastic. If the latter method is used it is possible to protect the viewing surface of small blocks from scratches by lowering a glass coverslip on to it while the spray layer is still liquid. We have used this method with success for small blocks containing sectioned teeth.
Other devices we have employed for protecting small specimens, or groups of specimens such as a developmental series of chick embryos, is to embed and retain the hardened block in a petri dish. This ensures, for small blocks that will receive a great deal of handling, microscopic viewing surfaces that are relatively free from scratches, and provides also for more or less dust-free storage. Our larger blocks are stored in plastic bags (Fig. 6).
We have found plastic letters pressed on to the surface of the block very suitable for labelling museum and special demonstration specimens. After labelling, the page 8surface is sprayed with plastic. Thin card labels sealed at one end of the plastic bag (Fig. 6) provide a more rapid and economical method for labelling specimens for a large class.
The paragraph below, outlining the procedures for embedding a small wet-preserved, soft-bodied specimen is given as a summary of the major techniques described above.
|(1)||Wash the specimen free from preservative.|
|(2)||Dehydrate first in 50% acetone. Place the specimen under vacuum.|
|(3)||Put the specimen in acetone under vacuum.|
|(4)||Clear the specimen by immersing it in styrine monomer. Place the specimen under vacuum if necessary.|
|(5)||Prepare a mould of suitable dimensions to allow at least ¼" of plastic all round the specimen. Caulk the joints with silicone wax and/or plasticine. Put a very thin coating of silicone wax on the floor and walls of the mould.|
|(6)||Warm a glass container in the oven at 50°C in readiness to receive a mixture of resin and catalyst.|
|(7)||Stir together gently enough resin and catalyst to give a ¼" layer of resin in the mould (the amount of catalyst to resin is 0.5 ml to 50 ml). Put the mixture into the oven at 50°C for 3-5 minutes. Stir gently and then pour into the mould.|
|(8)||Place the mould and mixture into the oven for 10-15 minutes and then allow the plastic to harden in the air until the suface is tacky.|
|(9)||Pour another layer of catalyzed resin prepared as in (7).|
|(10)||Place the specimen on the surface of this layer and allow it to sink under its own weight into this layer. Be careful not to trap air bubbles below the specimen.|
|(11)||Continue preparing, pouring, and hardening layers of plastic until the specimen is covered by ¼" of plastic.|
|(12)||Allow the plastic block to harden thoroughly.|
|(13)||Polish the specimen with varying grades of carborundum, then buff it with Brasso and finally Silvo.|
We wish to thank Mr. B. Perham, of A. R. Hatrick Ltd., for his helpful comments on plastic embedding techniques; and Mr. M. Loper, Technical Officer of the Zoology Department, V.U.W., for taking the photographs for the text-figures.