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

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determination of the position of the shell, facilitating
comparison. Protoconchs should be shown in apical view, at
right angles to the coiling axis or parallel to the coiling axis
with the transition proto- to teleoconch in the centre.
Bivalves should be shown with the outline of the shell
parallel to the image plane and the prodissoconch (with at
least long axis parallel to image plane) should be shown in
umbonal view.
SEM preparation of animals
External anatomy can provide many useful features. The
animal should be preserved in as natural a state as possible,
ideally following relaxation. In general, formalin or
glutaraldehyde fixed animals dry better than those fixed in
ethanol only. Additional post-fixation may be carried out
with osmium tetroxide (OsO 4 ), particularly if specimens are
viewed in older SEMs that necessitate high accelerating
voltages as OsO 4 will add conductivity to the specimen (see
Appendix 1). Post-fixation is not necessary for more modern,
low voltage or variable pressure/environmental SEMs.
Whether low voltage operation (< 1 kV), possibly with
increased spot size, or variable pressure/environmental mode
gives better results depends on the particular model of SEM
and the particular specimen and it is worthwhile
experimenting with various settings (see also above). Much
information can be obtained (Fig. 9), even using older SEMs
and crudely preserved (‘vodka’) specimens, with the results
usually surpassing visual examination under a
stereomicroscope.
Preliminary inspection
Wet specimens preserved in glass tubes do not need to
be removed from the tube for a quick assessment. The glass
tube can be immersed completely in the same preservative
and the distortion caused by the glass will disappear. An air
bubble in the container will also greatly reduce the distortion.
For quick approaches suitable for common and
abundant species, see ‘Removing the shell the fast way’. If
the specimen is rare or unique as much information as
possible should be obtained from it, including SEM of the
GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27
shell, information on external animal morphology etc.,
before removing the radula or other relevant internal
structures. The following techniques require some
experience and practice with common species is
recommended.
It is generally easier to extract bodies from dried
gastropod and bivalve specimens that have been rehydrated
as opposed to continuously fluid preserved specimens. In the
latter, the body is more firmly attached to the shell and it may
even be useful to dry the specimen and then soak it if other
methods are unsuccessful.
Limpets
Bodies can usually be separated from the shell, or dried
in the shell (see below: Tissue preparation for SEM), which
will protect the body from any damage when physically
removing the body from the shell. The shell with body can be
scanned or the body can be separated from the shell when it
has been dried. To separate the body from the shell with
minimal damage use a micro-scalpel or a piece of razor
blade, not a needle. After SEM documentation, the body can
be rehydrated in water for radula extraction.
Coiled gastropods
Because dried bodies are difficult to remove, the shell
should be photographed (SEM or standard
macrophotography) before trying to remove the body in case
the shell is damaged. Tightly coiled species are more difficult
than those with few whorls and a large aperture. From those
with a large aperture, the body can usually be extracted with
a fine needle with a small hook by inserting it at the
columellar side after rehydration in weak buffered formalin
and traces of a neutral detergent. Rehydration may take an
hour to a couple of days depending on the condition of the
body and the size. To speed up rehydration, evacuate the air
with a vacuum pump; use a small container to avoid
implosion by the glass breaking. The water may suddenly
start boiling when the pressure drops. A regular 20 ml glass
jar with a rubber stopper, connected to a water-jet pump with
a transparent polyethylene hose works well. In this way airbubbles
trapped inside the shell are replaced with water.
FIGURE 8. Different approaches to visualize surface texture using SEM as shown on a fossil micromollusc kindly made available by Mike
Vendrasco and Christine Fernandez (phosphatic internal mold of Mellopegma sp., Middle Cambrian, Georgina Basin, Australia). All images
were taken on a Zeiss EVO40XVP with an accelerating voltage of 20 kV and rather high probe current of 300 pA at a working distance of
10 mm. Scale bar = 100 µm. A. Secondary electron detector (SED) with bias of +300 V. This is the usual operating condition of the SED. B.
SED with bias of ±0 V. C. Secondary electron detector with bias of -50 V. All secondary electrons are repelled, and the SED operates as a
backscatter detector. D. SED with bias of -250 V. SED operating as a backscatter detector, excluding the secondary electrons as well as the
lower energy backscatter electrons. E. All four quadrants of QBSD. Macroscopic relief is completely obscured, and only microscopic
irregularities are visible. F. Single quadrant of four quadrant scintillating backscatter detector (QBSD). It shows slightly more macroscopic
contrast than G, but also has a slightly lower signal to noise ratio as shown by the somewhat more granular image. G. Two adjacent quadrants
of QBSD with normal polarity producing positive image. H. Two adjacent quadrants (opposite ones from G) of QBSD with inverted polarity
producing negative image. I. G and H combined. Notice fine detail of surface is best visible in A and E. The macroscopic undulations of the
shell are best seen with the SED used as a backscatter detector (C and D), The combination of normal polarity and inverted polarity signals
from the QBSD highlights the macroscopic undulations, while smoothing the minor surface irregularities. Images DLG.