Working at Higher Resolution
The method proposed here is mostly in use for higher Diptera, but
I don't see a reason why not to apply it to the lower ones, too.
In principle haemolymph and the internal soft parts are
transformed by KOH into a sort of soap, which is washed out by
glacial acetic acid. Dissection, inspection, and photography are
done in glycerol. One advantage of this method is, that no
poisonous substances are involved.
The chemicals needed are: 10% KOH, pure water, glacial acetic acid (99%), 2-propanol, glycerol. They should be available in your local pharmacy.
To be able to work without a coverslip in glycerol, check the
working distance of the objectives of your microscope. It should
be at least 2mm. The usual objectives with 20x and 40x
magnification have a far smaller working distance and you'll have
to buy objectives from an inverse microscope. If you plan to buy a
new microscope, the minimum requirement should be, that such
objectives are available.
Furthermore I use a set of watch glasses, Dumont forceps,
dissection pins, and an acute scalpel.
Point 3: "Overnight" is just a typical time, for Chironomidae an
hour can be enough, Bibionidae I'd usually clear, dissect, and
clear again. From the other two clearing chemicals I've seen, I
found lactic acid comparable, but didn't check it thoroughly (try
it), and Kreosote is carcinogenous. For using Euparal see below.
Points 7-10: The most important reason for bad resolution in
glycerol is, that one doesn't wait long enough for homogenization.
A typical value is 1 hour, but for large parts it can be much
longer. And you have possibly to wait again after every dissection
step. Bear in mind, that even if you don't see any streaks under
the comparably low resolution of the binocular, they can still be
strong enough to degrade the image in a compound microscope. The
second reason is, that the parts are floating around, or, if you
want to show a part in an upright orientation, slowly topple over.
This movement is mostly too slow to be observable under the
binocular, but in a series of exposures to be used for focus
stacking, it is clearly visible. Mostly this oblique stack can be
corrected in the same way as the stack resulting from a binocular.
Here is an example.
The highly diverse structures of the terminalia among Diptera
makes it impossible to give a unified howto of dissection. The
first two examples below show the typical brachyceran condition
consisting of epandrium, hypandrium and appendages. The next two
show the typical nematoceran forceps type. And finally I show two
examples of the many groups, which differ from the two basic
The last point to mention is permanent embedding of parts on a
slide to be able to use objectives with a higher resolution. In
some groups like Chironimidae, Phoridae, Sciaridae, specimens are
embedded immediately, even before inspection. In other groups,
mostly calytrate flies, the experts avoid embedding at all. Thus,
at first sight it looks like embedding is appropriate in some
groups and inappropriate in others. But obviously embedding has
the same advantages and disadvantages in all groups. If you embed,
you might miss characters, because after the sample is dried, the
side view of the parts is lost. If you don't embed, you might miss
characters, because some of them only become visible at higher
resolution in the embedding. Below I try to show a way through
this dilemma by discussing some pros and cons, present different
embedding media (and methods), and show some comparisons between
exposures taken in glycerol and Malinol (artificial Canada Balsam,
But first let me continue my recipe above for embedding in
Point 12: Take care that the alcohol doesn't evaporate befor
you've added the mountant. This causes irreversible damage.
Sometimes I place an extra drop of alcohol on the slide, before I
transfer the part (e.g. for legs). As Malinol is strongly adhering
to all materials, I found it convenient to use tooth pickers to
control the drop size and to discard them immediately into a
closed bin. The small drop remaining on the pin after orienting
the part, I remove with soft toilet paper.
Point 15: At this point we'll notice that drop size is crucial.
As Malinol almost doesn't shrink, it is less risky that a too
small drop causes distortion of the parts. But a too large drop
can cause a very slow reorientation, and after a few days you must
correct it. Even worse, too large drops make the use of objectives
with higher resolution impossible, since Malinol has the same
refraction index as glass and high resolution objectives are
calculated for standard coverslips with 0.13-0.17 mm thickness.
The best resolution can be achived, if the amount of mountant is
just as large as necessary, but this might prevent us from showing
different orientations. If you want to reorient the parts, don't
use too small coverslips.
Please note, that for embedding in Malinol no poisonous chemicals
are needed. Xylene only comes into play, when we have to
redissolve a slide, see below.
The usual alternative to storing the dissection result on a slide
is to store them in "genitalia vials" in glycerol. A clever
alternative is to glue the parts on a piece of card and attach it
to the same pin as the fly (recipe from Michael Ackland). Bear
these methods in mind for the following pros and cons of
Comparison of Embedding Media:
DMHF (Dimethyl Hydantoin Formaldehyde):
Mostly used in the UK. Can be soaked in water in a few minutes, the other media need about a day. The surface hardens quickly, which makes orientation inconvenient.
Mostly useful to embed undissected material (terminalia of Chironomidae or Sciaridae), because you save the clearing step. The sample takes days - weeks until complete clearing is achieved. I stopped using it after I had trouble with redissolving, but this might have had different reasons. You should definitely try it.
Malinol (artificial Canada Balsam, chemically identical):
Only one disadvantage, it can only be redissolved in xylene (probably even better in benzene, but this is even more carcinogenous). Xylene shouldn't be used outside the flue of a chemical lab, especially not in living space. If you really have to redissolve a Malinol slide, here is short description. The two important advantages of Malinol are, that it is not shrinking (there's almost no risk of distortion) and you can show different views in the same embedding (see recipe, point 15)
Finally have a look to some
photos of the same object taken in glycerol and Malinol,
which should help to make a decision whether to accept the extra
work and to take the risk of having to redissolve.