Currently, maggots from forensic cases are only rarely mounted on microscope slides. Why this is I cannot say. In any event, the coroners and homicide detectives for whom this is written need to know little about the subject, save to say that a well-prepared slide is both the clearest way to examine a maggot, and the most permanent record of that examination. Such a slide will still be like new when everyone involved in the case has long departed for another world.
For this and some other reasons in every case involving maggots I presently try to stain and mount some--ten is a good number--for use as a permanent record. For me at least this seems natural. Many years ago as a master's student one summer I made over 3,000 slides of scale insects. Although I never hope to repeat the performance, I do regularly use slides in all my entomological work, and would be lost without them.
The more I look at maggots on slides the more I realize how variable are most of the characters currently used in their taxonomy, and how easily an error could arise from using the extant keys and descriptions with only one or two specimens, especially if these are examined while they are in alcohol. The purpose of the rest of this section is therefore to instruct forensic entomologists in the basics of slide preparation.
Insect fossils have been found in amber that is in excess of 40 million years old. In spite of their great age when these fossils are polished every detail of the insects inside can be seen. Although appearing miraculous and separate from ordinary life this amber is really a very common substance. It is the hardened resin of certain species of coniferous trees. The whole idea behind slide mounts is to duplicate and improve upon this fossilization process.
The chief and best substance for a permanent slide mount is Canada balsam. This substance is a resin derived from the Balsam Fir. For lab use, it is purified and powdered, and then sold either as is, or dissolved in xylene, an organic solvent. The balsam/xylene mix into which the insect is placed is initially liquid and syrupy, but becomes harder with time, because the xylene slowly evaporates with exposure to heat and air. This process leaves a solid mount that can be examined with a microscope.
But before the maggot is placed into the balsam, it should be prepared in various ways to make the mount clearer. Part of this preparation is to remove the thin film of air on the surface of the maggot's cuticle or skin, which would obscure the tiny parts which are necessary for identification. Part is to remove the water in the tissues and gut. Water will not mix with the balsam resin, and if left in place will form a whitish halo around the mount. And part is to make structures that are naturally opaque transparent, as these would obscure the outlines of any structures they might overlay. And last, part is to selectively color or stain parts of the insect to bring out the details of particular interest. Standard preparation is described in the following paragraphs. The various reagents mentioned are best held in so-called "micro" or USBPI watch glasses1.
1. Place the specimens in an aqueous solution of potassium hydroxide (KOH) at room temperature for several days. I usually add the maggots as a group to a 10% solution in a 4-dram vial. KOH is a very caustic base. This means it will dissolve or "burn" flesh, including your skin and eyes. The idea is that the solution will remove all the tissues from the exoskeleton of the maggots.
2. Remove the maggots to 70% ethyl alcohol [EtOH] for 10 to 15 minutes. This washes the KOH from them. 70% isopropyl alcohol works just as well as does EtOH. While the maggots are in the alcohol, pierce each one with a sharp pin (on one side) and then flatten it with a small spatula. This is to remove the remains of the body contents. It also helps facilitate replacement of liquids within the body cavity, in this and succeeding steps.
3. To stain the maggots, transfer them to Specimen Clearing Fluid and leave them there overnight. Several drops of Double Stain should have been added to the fluid. The formulae for Specimen Clearing Fluid and Double Stain are given at the end of these instructions. These reagents are also available pre-mixed from entomological supply houses2.
4. Transfer the maggots back to 70% EtOH for 10 to 15 minutes. This is to wash the Specimen Clearing Fluid/Stain from inside their cuticle. It also allows you to "fine tune" the staining process, that is, to get the cuticle or "skin" of the maggot to show exactly the degree of transparency you want. The maggots will be very darkly stained when placed in the EtOH, and will become lighter the longer they remain there. If after 15 minutes the desired degree of transparency has not been obtained, leave the specimen in the EtOH until it has. Once again, 70% isopropyl alcohol will work just as well as EtOH.
5. Transfer the maggots to 100% EtOH for 10 to 15 minutes. Absolute alcohol (200 proof) dehydrates the specimen, that is, removes all the water.
6. Transfer the maggots to clove oil for 10 to 15 minutes. Your aim here is to put the specimens into a solution that will mix well with xylene and balsam. Absolute alcohol does not mix well with balsam, even though it will not cloud the mount. Furthermore, it has a different refractive index from the balsam. Therefore, if a drop of 100% EtOH is enclosed by balsam, you can tell. Light will not pass through the two substances the same way. Clove oil has neither of these disadvantages. It has the same refractive index as balsam, and can be mixed with it in any proportions.
7. Each maggot should be mounted separately in the middle of a standard microscope slide. Put a large drop of balsam in the middle of the slide. You want enough balsam to cover the specimen but not so much that the cover slip will float. As with everything else a little practice will tell you the appropriate amount to use.
Position the maggot on its side in the drop. This position is ideal for examining the mouth hooks and anterior spiracles. Then lay a cover slip over the drop. I try to use circular cover slips instead of square ones. Circular slips allow the balsam to spread much more evenly. Whether the cover slip is circular or square, be sure to apply it from one side. This procedure will assure that no air bubbles get trapped in the mount. Invariably these bubbles will congregate just over the most important characters of the specimen. Sometimes the specimen is too thick for the cover slip to lie flat. To fix this problem, place three or four slivers of broken cover glass in the balsam drop around the specimen. These bits of glass will act as props. Or you can buy small plastic nodules designed specifically for this purpose.
8. Transfer the slides to a slide-dryer or drying oven at ca. 400 degrees C for about two weeks. If you leave them in the dryer longer the balsam will begin to cook and turn dark. If you remove them early and then store the slides on their side, the balsam will slowly drain off and ruin the mounts, and perhaps the storage box they are in as well.
This is perhaps the place to mention that the slides need to be examined with both a dissecting microscope AND a compound microscope. I have a dissecting scope with a high power of 80x, and I find it ideal for examining the spiracles, mouth hooks, and other large structures of maggots. But this magnification is not sufficient to clearly trace the distribution of the minute spines so important in maggot identification. For this work I use a compound scope with a high magnification of 900x. Eventually I hope to buy a phase-contrast microscope, although I must say that for the huge amount of money the added advantage is minimal. But never-the-less I am used to one of these scopes, and feel under-equipped with anything else. I spent thousands of hours peering through one in my scale insect work.
phenol (liquefied) 2 parts
glacial acetic acid 4 parts
distilled water 1 part
5 ml aqueous acid fuchsin
1Available from Thomas Scientific, 99 High Hill Road at I-295, Box 99, Swedesboro, NJ 08085-0099 (ph. 609-467-2000).
2Both specimen clearing fluid and double stain are available premixed from BioQuip Products, 17803 LaSalle Ave., Gardena, CA 90248-3602 (ph. 310-324-0620; FAX 310-324-7931).