EMBEDDING ENTOMOLOGICAL SPECIMENS IN AN IMPROVED POLYESTER RESIN

J. BURGMANS, Horticultural Research Centre, Levin. INTRODUCTION Over the past 15 years different types of plastics have been used f o r the embedding of biological specimens. Many of these suffered from the disadvantages of chemical instability, discolouration on drying o r shrinkage. The recent introduction of stable, clear, resins into New Zealand meant that a modified mounting medium, requiring little skill or special facilities, is now availabIe f o r use. The technique described here enables specimens to be embedded in perfectly transparent blocks of resin, allowing good visibility of specimens of all types. Fig. 1 illustrates the clarity of the resins. DESCRIPTION OF MATERIAL The embedding resin is a polyester-type made from pure ingredients to keep the colour pale.! Colour stabilisers have been incorporated. As purchased, the resin has a pale greenish colour but this completely disappears during hardening. The curing of a normal polyester resin necessitates the use of two materials, the promoter or accelerator, and the catalyst. Addition of the accelerator does not cause the resin to react a t once but normal accelerators tend to shorten shelf life materially. With this resin a n accelerator has been introduced which does not aff'ect the shelf life of the
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Obtainable from Consolidated Chemicals Ltd., P.O. Box 15-104, Auckland, and R. & E. Tingeg Ltd., 30 Manners Street, Wellington.

resin. I t also ensures a low temperature during curing, and a final pale colour. I t s use has enabled the volume of the catalyst methyl ethyl ketone peroxide t o be c u t to 0 . 5 % of t h a t of t h e resin. The low catalyst level f u r t h e r assists in the attainment of a low tempera t u r e cure in mass and so prevents thermal strain which can lead t o the development of cracks a t a later stage.

PREPARATION OF SPECIMENS An essential preparatory material is a bottle of styrene monomer f o r wetting t h e surface of the specimen t o eliminate a i r bubbles. Fleshy specimens or those which have been preserved in formalin must be dehydrated by a series of immersions in successively stronger mixtures of alcohol and water and then allowed t o dry on t h e surface. An alternative method (Notini, 1 9 6 1 & DuPont de Nemours, 1966) is t o dip t h e wet specimen into acetone, allow t o air dry, soak in styrene monomer f o r one hour, and finally leave to soak i n uncatalysed resin f o r several hours. A f t e r draining off most of the resin t h e specimen may be embedded. Air dried insects should be arranged i n their natural position immediately a f t e r killing. The duration of treatment will depend on the n a t u r e of t h e specimen; a smooth beetle will require only a f e w moments whereas a moth may need a s long a s one hour. If a n exhaust pump is available t h e application of vacuum will assist in removing all air bubbles f r o m even t h e most irregular surface. EMBEDDING TECHNIQUE The first requirement is a suitable mould and there a r e several ways f o r making these. A simple method i s t o line a small cardboard box with cellulose tape, taking care t o press down t h e tape into t h e corners. A box mould may also be made from a perspex sheet held together with cellulose t a p e around t h e outside edges. Alternatively a permanent mould can be made using a silicone rubber compound, or a n elastic vinyl compound.2 The l a t t e r has the advantages t h a t i t is less expensive than silicone rubber and can be semoulded again simply by melting. The resin can be mixed i n a disposable container and should be measured o u t most carefully. Though thorough mixing is essential t o obtain a uniform!^ cured block care should be taken n o t t o introduce a large number of airbubbles by stirring too vigorously. The best f o r m of stirrer is a wooden or metal strip having a square end. This allows t h e resin t o be scraped from the sides and bottom of t h e container. A f t e r mixing until no striations a r e visible, t h e mixture should be allowed t o stand f o r 5 minutes t o allow airbubbles t o rise t o t h e surface. Before pouring, these should be carefully skimmed off.
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Silastic 588 R.T.V. Silicone Rubber and Vinamoulding compound can be obtained from R. & E. Tingey Ltd., 30 Manners Street, Wellington. 62

Sufficient resin t o cover t h e bottom of t h e mould t o a depth of 8" should be poured in and t h e top of the mould immediately covered t o exclude dust. The remainder of t h e catalysed resin is placed in a refrigerator t o delay setting. Although i t is less convenient and r a t h e r more wasteful of material, better results a r e obtained by preparing separate quantities of freshly catalysed resin f o r each layer. In about half a n hour in a warm room the resin in t h e mould will have gelled and the specimen can be arranged on the sticky surface. The resin or wetting fluid should n o t be allowed t o come into contact with the fingers; forceps should be used. The setting of t h e first layer of resin may be speeded up by placing t h e mould in a vessel of warm water or by local heating with a desk lamp. Care must be taken not t o overheat the resin and under n o circumstances must t h e setting of the resin be allowed t o progress beyond t h e soft gel stage. Pouring of t h e succeeding layers of resin must proceed before t h e preceding ones have fully hardened. If this precaution is not taken, strong refraction lines will be visible in t h e final block. Once t h e specimen has been a r ranged f u r t h e r resin should be poured into the mould, but only just enough t o hold the specimen in position. When this has gelled, t h e mould may be filled and l e f t t o harden overnight. The procedure is illustrated in Fig. 2. With some airdried insects i t is advantageous to use t h e vacuum technique, since a i r trapped in t h e dried out body has t h e habit of escaping in a chain of tiny bubbles. The vacuum technique is also advisable in t h e case of butterflies. Pre-wetting with styrene causes the loss of a i r space between t h e scales on the wings and in consequence, t h e white colours become transparent. One way of overcoming this is t o saturate t h e specimen with ether a n d embed immediately, before t h e ether has evaporated. A f t e r curing, t h e block is subjected t o strong pressure i n a book press or engineer's vice. When t h e correct amount of pressure has been applied, a n airspace will f o r m between t h e plastic and t h e wings. Because of t h e pressure applied, t h e plastic will pull away f r o m the specimen causing the original colours t o be restored. Considerable pressure can be applied without actually fracturing t h e block b u t a little experimenting is required before good results can be obtained. Space between the resin and t h e specimen is not desirable with embedded beetles b u t often occurs because t h e plastic does not adhere t o t h e hard, greasy chitin surface. In some cases airspace will develop all over t h e body and impair t h e view of t h e specimen. This f a u l t can often be prevented by heating t h e insect in a detergent solution in a water bath or by de-greasing with methylenechloride (Southgate 1953) before embedding.

Fig. 1. Vanessa itea and Porrhophele antipodiane embedded in resin.

Fig. 2. The block of resin is poured in three layers. Layer C is the supporting layer layer B anchors the specimen and layer A completes the block.

Before the top layer of resin is added a label may be inserted. Much of the value of plastic mounting lies in the information which can be included with the specimen. The following method of labelling has proved to be successful. 'Letraset' sheet, which is semi transparent, and has the letters of the alphabet printed on one side is the source of print. The letters can be transferred from this sheet onto the clear acetate film. If the surface of the acetate film is cleaned with alcohol before lettering, this helps to secure the small plastic letters more

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firmly. The lettered strip should now be dipped into catalysed resin, placed upon t h e surface of t h e previous layer and must be allowed t o harden. Unless this precaution is taken t h e plastic based letters will float off when t h e n e x t layer of plastic is applied. Alternatively these lettered strips can be dipped into the resin and allowed t o harden individually. Rate of cure is very dependent upon ambient temperature and if this is above 8 0 F , setting is so f a s t t h a t trapped airbubbles have no time t o escape. If i t is essential t o operate i n hot summer weather, t h e resin and mould should be pre-cooled in t h e refrigera t o r and the mould replaced f o r a t least 1 0 minutes a f t e r each pour. A f t e r two days a t room temperature t h e block will have become hard enough t o remove from the mould. This post-cure hardening can be speeded up by gentle heating b u t the temperature must not be allowed t o exceed 150F. F o r a block more than I" thick or of more than 1 cubic inch volume, heating is unwise a s it may lead t o shrinkage, warping, discolouration and t o t h e developm e n t of thermal strains. If exposed t h e surface of t h e block will remain sticky due t o t h e a i r inhibiting the cure. This can be prevented by covering t h e u p p e r surface with a piece of perspex when t h e resin has set. This sticky layer is very thin and could be removed by sanding lightly with No. 320 wet paper, followed by a treatment with No. 400 paper. The final polishing can be done by hand with a silicone car polish, or better still with a high speed mow. This treatment will give a n unimpaired view from all sides of t h e embedded specimen. If a coloured base is desired, this can be made by using "Epiglass" pigment pastes, which a r e available in black, white, red, green, blue a n d yellow. The pastes a r e very concentrated and only a small quantity is required. A stock solution of paste with alcohol can be prepared and added a s required t o t h e resin. SCOPE OF EMBEDDING TECHNIQUE Although this method of preserving is time consuming and may take several days t o complete, i t is relatively simple. If reasonable care is taken i t will give excellent results. There a r e a number of obvious applications of t h e technique, particularly in teaching and diagnostic work. Most insects a r e readily mounted. Mineral specimens or small air-dried insects a r e best used f o r initial work. With some fleshy specimens there is some danger of loss of colour a n d this is t r u e in t h e case of caterpillars (Notini, 1961). Embedding has been used t o preserve insect-damaged leaves and flowers. Colour slides can be included with the specimens, t o explain a certain insect cycle or damage.

ACKNOWLEDGEMENTS
I a m indebted f o r the assistance I received from Mr. D. Manson, Horticultural Research Laboratory, Levin, and f o r the technical advice f r o m Mr. P. B. H u n t , Chief Chemist, Consolidated Chemicals Ltd., Auckland. REFERENCES BJORKSTEN, J., TOVEY, H., HARKER, B. and HENNING, J., 1956: Polyesters a n d their applications. Reinhold Publishing Corporation. 430 P a r k Avenue, N.Y. DU PONT DE NEMOURS & CO., Plastics Department, Wilmington, Delaware, U.S.A. Private communication. I.C.I., PLASTICS DIVISION, Welwyn Garden City, Herts., U.K., 1966 : Acrylics f o r embedding. Technical Service Note G110. MEYDEN, A. F. Van Der, 1966: Embedding biological specimens in solid plastic. J. Soc. Audi. Visual Ed. N.Z. 1 (1). NOTINI, G., SIGRID v WETTSTEIN, 1961: Embedding biological objects i n plastic. Bulletin 36, Royal School of Forestry, Stockholm, Sweden. PLASTICS (London) 1 6 : 249-50 (1951). Use of polyester resins f o r embedding biological a n d archeological specimens. RANG1 CHEMICALS, P.O. Box 22-220, Auckland. Embedding specimens in methacrylate resins. 1962, Bulletin, Rohm a n d Haas Co. SOUTHGATE, B. J., 1953: A convenient method of degreasing insect specimens. Entomologist, 86.