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

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6 GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27 • Use some kind of rack or stand to keep them available and ready for use. • Micro-pipette tips used in molecular biology (100– 1000 µl) make excellent tip and needle protectors. • Preferably use tools made of material that will not deteriorate quickly in salt water (e.g., stainless steel). • For microscope work, a comfortable seat of the correct height is essential. For fine manipulation, steady your body by resting your elbows and wrists on the table, use the back support of your chair, and place your feet firmly on the ground. Consider breathing rhythm as it moves the ribcage and arms. • Keep equipment clean to avoid deterioration and contamination. There are a number of suppliers of suitable equipment who can also offer useful information (e.g., http://www. finescience.com; http://www.mccronemicroscopes.com). We do not illustrate most of the readily available tools, only those that are custom made or enlarged views ordinarily not shown (Fig. 4). Sieves For a more efficient examination of samples containing a large proportion of sediment, the residues should be divided into size fractions by using graded sieves (e.g., 10, 5, 2.5, 1.0 and 0.4 mm mesh size). If one wants all specimens, including larval shells, 100 µm mesh is needed, while for all adult species 0.4 mm is suitable. Fractions larger than 5 mm can be examined with the naked eye. For 5–2 mm a low power magnifier can be used, although a stereomicroscope is preferable and gives a better yield as untypical molluscs are more easily recognised. For smaller fractions a stereomicroscope is essential. Commercially made sieves and even shakers for banks of sieves are available, or screens can be constructed from various sizes of wire mesh (Fig. 4H). Sieves can also be made by using short pieces of PVCpipe, 50–250 mm diameter (Fig. 4G). A piece of metal (preferably stainless steel) mesh slightly wider than the pipe can be placed on a piece of aluminum foil on a hot plate and the pipe pushed down on it until the end of the pipe starts to melt. At that point, put it on a cold surface, still with some pressure, so the net does not separate from the soft plastic. Trim off any surplus net and grind the edge to remove any free wires. Instead of a net, a perforated sheet of stainless steel can be used. It is a little more difficult to work with but makes very sturdy sieves that are less readily clogged. For fieldwork, collapsible nets with a fine mesh (
TECHNIQUES FOR STUDYING SMALL MOLLUSCAN SPECIMENS 7 Microscope A good-quality dissecting microscope is essential. A minimum magnification of 50x is desirable. In general, those with stepped magnifications have better optical quality than those with zooms. For illumination, traditional focusable light sources, halogen fibre optic, or light emitting diode (LED) lights can be used. The traditional lights can often be more precisely positioned and a greater working distance can be achieved because the lights can be focused, unlike standard fibre optic lights, allowing more freedom to manipulate objects. On the other hand, fibre optics lights have a higher light output and optional focus attachments are available. LED lights are similar to fibre optics, though a little weaker. They are particularly useful for field work due to being lightweight and in having long-lasting bulbs. A substage illuminator, a tiltable mirror, or a dark-field base can be helpful when searching for radulae in maceration solution (see below). The base of the microscope can be mounted in a hole in the working platform (bench or desk), so that the surface area of the microscope is level with the remainder of the desk. Pipettes Pasteur pipettes of glass with a rubber bulb are useful; the diameter of the tip can be adjusted by cutting. Disposable Pasteur pipettes of polyethylene do not deteriorate and can easily be cut to tip diameters of up to 5–6 mm. Pipettors (Eppendorf and similar brands) used in molecular biology are considered by some to be bulky and difficult to manoeuvre when used under a microscope, while others like their precise flow control. There are also devices that produce a constant vacuum on a very small bore to pick up specimens which are released when the vacuum is broken (Hemleben et al. 1988). For a pipette to form very small droplets (e.g., for radular work), use a commercially available disposable pipette tip for µl work; insert it in a fitting polyethylene tube 50–80 mm long and seal the other end (Fig. 4E). The stiffness of the polyethylene tube gives better control over the quantity delivered. A ‘home-made’ capillary glass-tube can also be used as a tip. Forceps There are five main types of forceps used for work with micromolluscs. 1. Entomology forceps. Made of thin spring steel, they are available with a variety of tip designs, which can be further adapted using a sharpening stone. Some users find they have good handling properties with a reduced risk of breaking fragile specimens while others find that the tips do not meet exactly, or are too slippery. With inexperienced users, specimens, especially smoothshelled gastropods, can be catapulted some distance. 2. Watchmaker’s forceps are available with very fine tips. Many qualities and shapes of tips are available ranging from the expensive straight MORIA MC-40 model which has the finest tips currently available and is made of stainless steel. There are a wide range of similar, cheaper models available. Watchmaker’s forceps are most suited for anatomical and radular work, but with practice they can also be used for routine sorting, including handling fragile specimens. 3. So-called ‘Iris forceps’. These very soft forceps have a rather broad tip (for example, the MORIA MC-32 or 32B has a tip of ca 0.8 mm), which can be either smooth or serrated. These forceps are excellent for handling specimens 5–10 mm and smaller. The shape of the tip can be easily modified with a file or sharpening stone. They are almost as soft as entomology forceps, but less flimsy. 4. Stub handling forceps. There are two basic types for handling SEM stubs; one made for handling Cambridge stubs, by holding it in a track in the edge and another designed for gripping the pin of all 1/8” pin stubs. Grind the tips of the former so they become more slender for a less tight fit in the groove and of the latter to a finer point so they can be more easily inserted under the stub. The Cambridge stub forceps can be modified to have narrower and less curved tips. 5. Bamboo forceps (Fig. 4D). One of us (TS) makes forceps from two pieces of bamboo. The tips of the bamboo pieces are shaped with a knife and sand paper, which can be accomplished in a relatively short time. Bamboo is softer than steel and is suitable for manipulation of fragile specimens and anatomical manipulations. Microscissors Microscissors come in a variety of models and a wide price range. Spring loaded scissors are suitable in many instances, which can be complemented with a couple of cheap, slightly larger ones for standard work. For particularly delicate work, a pair of extra fine ones (e.g., MORIA extra fine) can be useful. They come with various tip configurations (pointed, blunt, angled) and are rather delicate and expensive. Scalpels Scalpels with a fixed blade are not recommended as they need re-sharpening, are expensive and corrode easily. The common types with a flat metal handle and disposable blades are suitable for most purposes. There are many different blades available; microsurgery scalpels (e.g., FST 10315-12) are excellent for opening very small bivalves and any other work where regular scalpels are too large. A cheap and very good alternative is to use a needle holder to hold a broken piece of razor blade (see below). Different brands of razor blades break in different ways. Pin and needle holders These come in different sizes and materials, some having a small chuck that will hold the finest needles. The handles vary (diameter, shape and texture) to suit different preferences and can be colour coded for easy identification of the various needles. Dismantle and clean the needle holder
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