Molluscan Research: Techniques for collecting, handling, preparing ...

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thoroughly, preferably overnight in an incubator. When
transferring radulae onto the cover slip, be careful to
prevent water getting under the cover slip as it will
unstick it.
The glass cover slip is released from the histo-slide base
by applying some water to the edge of the cover slip.
Capillary forces will pull the water under the cover slip
and dissolve the glue. The loose cover slip is then
mounted on a SEM stub with new glue.
• Rhipidoglossate (Vetigastropoda) and docoglossate
(Patellogastropoda, Polyplacophora) radulae have
overlapping lateral and marginal teeth, which obscure
other teeth. To mount these types of radulae, glue some
thin pieces of wire, or hair, or needles of a diameter
close to the width of the radula and cover them with
glue (Fig. 12A, B). Cover slips may also be prepared
with a series of wires etc. of different widths. Radulae
can then be mounted longitudinally, on top of these
wires with the marginal teeth bent outwards and
downwards. This affords a better view than mounting
radulae across the wire (e.g., Strasoldo 1991), but
requires some practice. Breaking up a flat-mounted
radula will also give the necessary data.
The orientation of a mounted radula can be doublechecked
under a light microscope with a 25 or 40x objective
although care is needed as the working distance is only 1–0.2
mm, depending on the lens. For radulae mounted on glass
slides, commonly available transmitted light microscopes
can be employed. Stub mounted radulae can be checked with
compound microscopes equipped for (or improvised) epiillumination
or with high power stereomicroscopes.
A radula that has been accidentally improperly mounted
can often be released from the mounting surface.
• For PVA glue, add water with a paint brush or a fine
pipette, soak the radula, remove and remount it. Do not
disturb the glue excessively, as it may invade the radula.
• If mounted on double-sided carbon tabs or tape, a
radula can be released by soaking it in a large drop of
water for a minute and peeling it off the carbon tab from
one end. Then, the radula may be remounted and dried
again. Once the radula has been sputter coated and
viewed in the SEM, the radula is more firmly stuck to
the carbon tab. Water will usually not be sufficient to
release the radula, but ethanol usually works. Coated
GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27
radulae are usually stiffer and more brittle than fresh
ones.
Very small specimens
The following variation of the method above has been
used for very small radulae (e.g., those of cimids with a
length of ca 60 µm) or post-larval gastropods. Soak the
specimen in a small quantity of distilled water: 1–6 drops
with a fine pipette in the depression slide or 1/3 or less of the
depth of the solid watch glass. Dissolve 1/4 of a tablet of
KOH to 50 µl (= 1–2 drops) of water. The tablets can be
readily split using a small stainless wire cutter. Heat the
solution in the incubator at 50°C for 15 minutes or a little
more. Usually the specimen will not dissolve but remain in a
lump of clarified tissues. Transfer the lump with a needle
(bent 90°), or a pair of forceps (an inferior method because
more fluid is transferred) to a drop of distilled water on a
cover-slip. Usually the lump of tissue will dissolve in a
fraction of a second. Add a drop of distilled water, close to,
but separated from, the point where the specimen was
dissolved and pull the radula over to this without allowing
the two drops to merge. Remove the dirty water with a small
piece of lint free tissue curled around the tips of a pair of
forceps and pinched in position by a little rubber band
around the upper part of the forceps. Wash the radula a
couple of times more—the radula should now be in very
clean water.
Manipulation of radula
Manipulation techniques vary with the mounting
surface chosen. Mark the position of the radulae, because
they are often easier to spot with a light microscope than in
the SEM.
For carbon tabs, the radula can be manipulated in a
small (preferably distilled) water drop using a pair of fine
tungsten needles. The water will evaporate at room
temperature in one to two minutes (ethanol evaporates too
fast for many small radulae, although it works very well with
larger radulae and has less surface tension than water [but see
remarks on some vetigastropod radulae above]). The surface
tension of the water will help in flattening the radula along
its long axis. At the point when the radula is still moist,
but when there is no free water around the radula (a period of
about two to three seconds), gently spread the radula out with
the needles. The outer rows of radular teeth tend to fold
over the central field when they dry, therefore it is important
FIGURE 12. A, B. Radulae mounted on long axis of pins. Scale bar = 2 mm. B, enlarged view of A. Scale bar = 200 µm. C. Assorted radulae
mounted on double sticky tape/carbon tab. Scale bar = 2 mm. D. Radula of Scissurella mirifica (A. Adams, 1862) showing tear in centre. Scale
bar = 200 µm. E. Enlargement of D showing full exposure of base of lateral and marginal teeth. Scale bar = 20 µm. F. Same radula as D. in
area not torn; note less exposure of the base of the teeth. Scale bar = 20 µm. G. Dikoleps nitens (Philippi, 1844) [Skeneidae] juvenile. Scale bar
= 10 µm. H. Dikoleps nitens adult, approximately 1 mm shell diameter. Scale bar = 10 µm. I-K. Haliotis discus hannai Ino, 1953 seven days
old larvae from a culture in Japan. I. Entire radula. Scale bar = 10 µm. J. Full width of row enlarged. Scale bar = 10 µm. K. Larval shell of
animal from which radula was obtained. Scale bar = 100 µm. Images: A–C, G–K: AW; D–F: DLG.