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EMBEDDING ENTOMOLOGICAL SPECIMENS INAN IMPROVED POLYESTER RESINJ. BURGMANS,Horticultural Research Centre, Levin.

INTRODUCTIONOver the past 15 years different types of plastics have beenused f o r the embedding of biological specimens. Many of thesesuffered from the disadvantages of chemical instability, discolour-ation on drying or shrinkage. The recent introduction of st able,clear, resins into New Zealand meant that a modified mountingmedium, requiring little skill or special facilities, is now availabIefor use. The technique described here enables specimens to be em-bedded in perfectly transparent blocks of resin, allowing good vis-ibility of specimens of all types. Fig. 1 illustrates the clarity ofthe resins.DESCRIPTION OF MATERIALThe embedding resin is a polyester-type made from pure in-gredients to keep the colour pale.! Colour stabilisers have been in-corporated. As purchased, the resin has a pale greenish colour butthis completely disappears during hardening. The curing of a nor-mal polyester resin necessitates the use of two materials, the pro-moter or accelerator, and the catalyst. Addition of th e acceler-at or does not cause the resin t o react a t once but normal acceler-ators tend to shorten shelf life materially. With this resin an accel-erator has been introduced which does not aff'ect the shelf life of th e

1 Obtainable fr om Consolidated Chemicals Ltd., P.O. Box 15-104,Auckland, and R. & E. Tingeg Ltd., 30 Manners St reet , Wellington.

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resin. I t also ensures a low tem per atu re during curing, and a finalpale colour. It s use has enabled the volume of t he catalyst methylethyl ketone peroxide to be cut to 0.5% of t ha t of th e resin. Thelow catalyst level further assists in the attainment of a low temper-at ure cure in mass and so prevents thermal s train which can leadto the development of cracks a t a lat er stage.

P R E P A R A T I O N OF S P E C I M E N SAn essential preparato ry material is a bottle of st yrene mon-

omer for wetting th e surface of the specimen to eliminate a irbubbles.

Fleshy specimens or those which have been preserved in forma-lin must be dehydrated by a series of immersions in successivelystronger mixtures of alcohol and water and then allowed to dry onth e surface. An alternati ve method (Notini, 1961 & DuPont deNemours, 1966) is to dip the wet specimen into acetone, allow toair dry, soak in styrene monomer for one hour, and finally leave tosoak in uncatalysed resin fo r several hours. After draining off mostof the resin the specimen may be embedded. Air dried insects shouldbe arranged in their natural position immediately after killing.

The duration of treatment will depend on the nature of thespecimen; a smooth beetle will require only a few moments whereasa moth may need a s long as one hour. If an exhaust pump is avail-able the application of vacuum will assist in removing all air bubblesfrom even the most irregular surface.

E M B E D D I N G T E C H N I Q U EThe first requirement is a suitable mould and there are several

ways for making these. A simple method is to line a small card-board box with cellulose tape, taking care to press down the tapeinto the corners. A box mould may also be made from a perspexsheet held together with cellulose tape around the outside edges.Alternatively a per manent mould can be made using a siliconerubber compound, or an elastic vinyl compound.2 The lat ter has theadvantages that it is less expensive than silicone rubber and can besemoulded again simply by melting.

The resin can be mixed in a disposable container and should bemeasured ou t most carefully. Though thorough mixing is essentialto obtain a uniform!^ cured block care should be taken not to in-troduce a l arg e num ber of airbubbles by st irring too vigorously.The best form of s tirrer is a wooden or metal strip having a squareend. This allows th e resin to be scraped from the sides and bottomof th e container. Aft er mixing until no striations ar e visible, th emixture should be allowed to stand for 5 minutes to allow air-bubbles t o rise to th e surface. Before pouring, these should becarefully skimmed off.2 Silastic 588 R.T.V. Silicone Rub ber and Vinamoulding compoundcan be obtained from R. & E. Tingey Ltd., 30 Manners Street,Wellington.

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Sufficient resin to cover the bottom of the mould to a depthof 8" should be poured in and the top of the mould immediatelycovered to exclude dust. Th e remaind er of the catalysed resin isplaced in a refr ige rato r to delay setting. Although i t is less con-venient and ra the r more wasteful of material, better results ar e ob-tained by preparing separate quantities of freshly catalysed resinfo r each layer. In abo ut half a n hour in a warm room the resinin the mould will have gelled and the specimen can be arranged onth e sticky surface. The resin or we tti ng fluid should no t be allowedto come into contact with the fingers; forceps should be used.

The se ttin g of th e first layer of resin m ay be speeded up byplacing th e mould in a vessel of wa rm wa te r o r by local heatingwith a desk lamp. Care must be taken not to overheat the resin andunder n o circumstances must t he s etti ng of the resin be allowed toprogress beyond th e soft gel stage. Pouring of t he succeedinglayers of resin must proceed before the preceding ones have fullyhardened. If this precaution is not taken, strong refraction lineswill be visible in th e final block. Once th e specimen has been a r-ranged further resin should be poured into the mould, but only justenou gh to hold th e specimen in position. Whe n this has gelled, th emould may be filled an d le ft to ha rd en overnight. The procedure isillustrated in Fig. 2.

With some airdried insects it is advantageous to use th e vacuumtechnique, since air trapped in the dried out body has the habit ofescaping in a chain of tin y bubbles. The vacuum technique is alsoadvisable in th e case of butterflies. Pre-wet ting with sty re ne causesth e loss of ai r space between th e scales on th e wings and in conse-quence, th e white colours become trans pare nt. One way of over-coming this is to saturate the specimen with ether and embed im-mediately, before the ether has evaporated.After curing, the block is subjected to strong pressure in abook press or engineer's vice. When the correct amount of pressurehas been applied, an airspace will form between the plastic and thewings. Because of t he pressure applied, th e plastic will pull awayfr om the specimen causing the original colours to be restored. Con-siderable pressure can be applied without actually fracturing theblock but a little experimenting is required before good results canbe obtained.

Space between the resin and the specimen is not desirable withembedded beetles but often occurs because the plastic does not ad-here to th e hard , greasy chitin surfac e. In some cases airspace willdevelop all over th e body an d impa ir th e view of t he specimen.

This fault can often be prevented by heating the insect in adetergent solution in a wa ter bath or by de-greasing with methylene-chloride (Southgate 1953) before embedding.

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Fig. 1. Vanessa iteaandPorrhophele antipodianeembedded 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 beincluded with the specimen. The following method of labelling has proved to besuccessful.

'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 st rip should now be dipped into catalysed resin,placed upon the surface of the previous layer and must be allowedto harden. Unless this precaut ion is taken t he plastic based lett erswill float off when the next layer of plastic is applied.

Alternatively these lettered strips can be dipped into the resinand allowed to harden individually.

Rate of cure is very dependent upon ambient tempe ratur e andif this is above 80 F , sett ing is so fa st th at trapped airbubbles haveno time to escape. If i t is essential to operate in hot summerweather, the resin and mould should be pre-cooled in the refriger-ato r and the mould replaced fo r a t least 10 minutes af te r eachpour. Af te r two days a t room temp eratu re th e block will havebecome har d enough to remove from the mould. This post-curehardening can be speeded up by gentle heating but the temperaturemust not be allowed to exceed 150F. For a block more than I"thick or of more than 1 cubic inch volume, heating is unwise a s itmay lead to shrinkage, warping, discolouration and to th e develop-ment of thermal strains.

If exposed t he su rfa ce of t he block will remain sticky due t oth e ai r inhibiting the cure. This can be prevented by covering th eup pe r sur fac e with a piece of perspex when th e resin has set. Thissticky layer is very thin and could be removed by sanding lightlywith No. 320 wet paper, followed by a treatment with No. 400paper. The final polishing can be done by hand with a silicone ca rpolish, or bet te r still with a high speed mow. This trea tmen t willgive a n unimpaired view from all sides of th e embedded specimen.

If a coloured base is desired, this can be made by using "Epi-glass" pigmen t pastes, which ar e available in black, white, red, green,blue an d yellow. The pastes ar e very concentrated and only a smallquantity is required.

A stock solution of paste with alcohol can be prepared andadded a s required t o th e resin.

S C O P E O F E M B E D D I N G T E C H N I Q U EAlthough this method of preserving is time consuming and may

take several days to complete, it is relatively simple. If reasonablecare is taken it will give excellent results.

The re ar e a number of obvious applications of th e technique,particularly in teaching and diagnostic work.Most insects are readily mounted. Mineral specimens or smallair-dried insects are best used for initial work.

With some fleshy specimens there is some danger of loss ofcolour an d this is tr ue in th e case of caterpi llars (Notini, 1961).Embedding has been used to preserve insect-damaged leaves andflowers. Colour slides can be included with the specimens, to ex-plain a certain insect cycle or damage.

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ACKNOWLEDGEMENTSI am indebted for the assistance I received from Mr. D. Man-

son, Horticultural Research Laboratory, Levin, and for the tech-nical advice from Mr. P. B. Hu nt , Chief Chemist, ConsolidatedChemicals Ltd., Auckland.

REFERENCESBJORKSTEN, J., TOVEY, H., HARKER, B. and HENNING, J.,

1956: Polyesters and their applications. Reinhold Pub-lishing Corporation. 430 Par k Avenue, N.Y.

DU PONT DE NEMOURS & CO., Plastics Department, Wilmington,Delaware, U.S.A. Pr iva te communication.

I.C.I., PLA STICS DIVISION, Welwyn Garden City, Herts., U.K.,1966: Acrylics fo r embedding. Technical Service NoteG110.

MEYDEN, A. F. Van Der, 1966: Embedding biological specimens insolid plastic. J. Soc. Audi. Visual Ed. N.Z. 1 (1).

NOTINI, G., SIGRID v WETTSTEIN, 1961: Embedding biologicalobjec ts in plastic. Bulletin 36, Royal School of Forest ry,Stockholm, Sweden.

PLASTICS (London) 1 6: 249-50 (19 51). Use of polyester resinsfor embedding biological and archeological specimens.

RANG1 CHEMICALS, P.O. Box 22-220, Auckland . Embeddingspecimens in methacryl ate resins. 1962, Bulletin, Rohmand Haas Co.

SOUTHGATE, B. J., 1953: A convenient method of degreasing in-sect specimens. Entomologist, 86.