Slide mounting in Balsam
The temporary nature of Hoyer's, or other similar media, makes it necessary to use other more permanent mountants for important specimens such as types, potential types or even voucher specimens. Most chalcidoid taxonomists prefer the use of Canada balsam to other media, such as Euparal, because it is easier to work with when mounting dissected specimens and also because its permanence is proven. It is also easily soluble in xylene after very many years thus making it possible to remount poorly mounted specimens. Use of balsam also makes it possible to dissect specimens and mount the various parts in such a way that taxonomic characters can bee seen more clearly. This particularly applies to antennal and facial characters.
Many taxonomists have avoided the use of balsam in favour of Hoyer's because good slides are more difficult and take longer to prepare. A further, very valid criticism is that the refractive index of balsam is very close to that of chitin and thus many cuticular structures, such as shallow sculpture, can be very difficult to make out. This can be overcome largely by the use of a phase contrast or refraction microscope. Other difficulties encountered, eg loss of pigmentation, crumpling of wings, etc., can be avoided by use of the following technique which can be used for making slides of even the smallest specimens.
A method describing mounting specimens whole under one coverslip is described by Platner et al. (1999). The following method requires that different parts of the body are placed in four or five different positions on the slide (Fig. 6, below) . These are best turned through 180º to allow for inversion of the image when using a compound microscope. It is also advisable to place each part in exactly the same position on the slide since this will facilitate subsequent examination and comparison of specimens. It helps to draw the outline of a slide on a flat card and to mark the required position of each part on a card. As each part of the insect is put onto the slide, its position can be marked by holding the slide in position within the outline and placing a drop of balsam in the required position on the slide.
|Fig. 6 Positions of different body parts mounted in balsam on slide.|
Before proceeding further, it is essential that the balsam to be used is of the correct consistency. For best results use natural, filtered, Canada balsam, but if this is not available then balsam in xylene, eg toluene, since this may result in the balsam "going off" too rapidly making the dissecting out of mouthparts, genitalia etc. very difficult. The balsam should be very thick and just forming strings when withdrawn on the head of a pin. It should be possible to invert a half-full 50 x 25 mm tube of balsam for 1-2 s without the balsam running to the top.
(1) Mount the specimen on a card so that the wings are completely free of adhesive (see above).
(2) When the specimen is completely dry remove wings with a fine pin and place in a drop of balsam on a slide. Dilute this balsam with xylene if necessary and arrange wings symmetrically with a fine pin. [Specimens that have previously been Critical-Point Dried may have air trapped in the wing veins that will not be removed by this method. To prevent this, before transferring the wings to balsam on a slide place them in 95% alcohol in a solid watchglass for about 10 minutes and cover with a cover glass. Next add two drops of clove oil and leave off the coverglass to allow the alcohol to evaporate leaving the wings in clove oil alone. Leave for a few hours and then transfer to a drop of balsam on a slide as described.] .
(3) Soak specimen off card and place in a 10% solution of KOH (potassium hydroxide) in a small 25x12mm glass tube.
(4) Either place the tube on a hot plate or treat as described in (5) below. The best results for clearing in KOH are achieved by putting the tube containing the KOH pus specimen to be cleared on a hot plate with a surface temperature of about 93-100°C. After a time the tube should examined. At 93°C smaller, paler, specimens should require 35 minutes for clearing whilst larger, darker specimens will require no more than 40 minutes. The exact timing can be found by experimentation. Tubes placed in a recess block at the same temperature may require only 5 minutes because the temperatures attained by the KOH will be higher. It has been found that material cleared on a got plate in this manner retain their colour better and are less likely to collapse when finally transferred to balsam that using the method outlined in (5) below.
(5) If the specimen has never been in alcohol then leave at room temperature (20ºC) for 48 h. However, if the specimen has been in alcohol at any time leave at room temperature for 72 h or at room temperature for 24 h and then for a further 24 h at 40ºC.
This timing is totally irrespective of size and colour. In other words a small, yellow Aphytis requires the same treatment as a large, dark Trichomasthus.
It is possible to leave specimens in KOH for a slightly shorter length of time, but this will increase the likelihood of the antennae or head collapsing when the specimen is finally transferred into balsam.
(6) Using a teat pipette transfer the specimens to glacial acetic acid in a solid watchglass.
After this and each of the following five steps the watchglass must be covered with a lid.
(7) After 10 min, remove glacial acetic acid and replace with 5 drops of distilled water.
(8) After 10 min, add 5 drops of 70% alcohol.
(9) After 10 min, remove liquid (35% alcohol) and replace with fresh 70%
(10) After 10 min, add 5 drops of absolute (or 95%) alcohol.
(11) After 10 min, remove liquid (80-85% alcohol) and replace with at least
four drops of absolute alcohol, enough to cover the specimen.
(12) After 10 min, add 3 drops of clove oil (or cedar wood oil) and leave cover off watchglass
(13) After about 20 min, transfer the body to a small drop of balsam on the slide separate the head from the prothorax. Transfer the headless body back to the solid watchglass.
(14) If required, dissect out and arrange the mouth parts by positioning the head face downwards. Hold in place with a fine needle and separate the labiomaxillary complex ( and labrum if possible) intact with fine micro-pin. Open out the madibles with a pin and at the same time rotate at least one so that the teeth are clearly visible (it should be possible for the mandible to be held in position by use of the remaining ligaments or by its condyle.) Try to leave mandibles attached to head or they may subsequently
"swim" in the balsam after the coverslip has been added. Turn the head back so that it is now flat on the occiput.
(15) Separate the antennae from the toruli using the point of a fine minuten pin.
(16) Transfer the antennae to another drop of balsam and position as required.
(17) Position the head and labio-maxillary complex as desired.
(18) Transfer the thorax and gaster to a drop of balsam on the slide.
(19) Arrange the specimen with its dorsal surface uppermost and the legs well displayed.
It may be necessary to mount the male genitalia or ovipositor complex separately, eg Encyrtidae.
Before arranging thorax remove relevant parts and treat as follows.
(20) Place slide in a dustproof container and place this in an oven at 40ºC for at least 2-3 weeks to let the balsam dry hard. [This part of the process can be speeded up by placing the slide on a hot plate at 100°C for three hours.]
(21) Remove slide from oven.
(22) Place some balsam on the head and thorax in sufficient quantity that when a 6 or 7 mm coverslip is placed on this, and pushed downwards until the balsam spreads to fill the coverslip, there will be only a very small space between the specimen and the coverslip.
The correct amount of balsam is essential: too much and the head or thorax may start "swimming" after a while or it may be impossible to focus the microscope on the lowermost structures because the objective of the microscope will hit the coverslip; too little and he head or thorax may be crushed as the balsam dries.
The correct amount of balsam to use is best learnt by experience, but it may be aided as follows:
(23) Place a 6 or 7 mm coverslip on the blob of balsam, keeping it as level as possible, and gently push it downwards until the balsam has spread completely to the edge of the coversleip.
(24) Check that the coverslip is horizontal in relation to the slide by holding the slide up at eye level. If raised on one side then gently push downwards, and very slightly outwards, on the side until the coverslip is level.
(25) Place a drop of balsam on each of the remaining uncovered parts and put on a coverslip. The amount of balsam used should be the minimum quantity required to completely fill the area under each coverslip when in place.
If necessary, the balsam used in covering the wings and gaster (if dissected separately) may be thinned with xylene. This should be avoided with antennae because it may result in them being crushed slightly as the balsam dries.
Separating the head from the thorax and antennae from the head without a lot of practice can be very difficult to accomplish without damaging the specimen. A slightly easier method has been described by Noyes (1990). This entails knocking the antennae of the head and the head off the thorax whilst the dry specimen is still on the card, before soaking it off and transferring it to KOH. These parts are then attached to the thorax using a minute blob of balsam (softened by xylene vapour emanating from the nearby head of a pin that has been dipped in xylene) and remain attached until the specimen is finally transferred to clove oil.