Simple methods for morphological observation of nematodes

Abstract

Some quick and easy techniques for nematode morpho-logical observations are provided. Silicone grease or petro-leum jelly can be applied to adjust the nematode direction to observe lateral or ventral aspects. Small spots of the grease are placed between the slide glass and coverslip to fix the coverslip and adjust sample thickness. Nematodes can be turned by moving the coverslip, which can be pushed to observe the complicated stomatal morphology. Squashing of the nematode is handy to evert the buccal cavity. Furthermore, diluted detergents (0.1-0.5% Tween 20 or 80) can be used to remove bacterial particles stuck on the nematode surface and observe the surface structure of the nematode. Despite their small body size, nematode morphology is very complicated. Partly because they are so small, some characters are difficult to interpret correctly. For example, the stomata of some diplogastrid nematodes are asymmet-ric and anisotopic, and close examination is required to understand its structure correctly. Baldwin et al. (1997) and Giblin-Davis et al. (2006) employed transmission electron microscopy (TEM) to interpret the stomatal structure of Acrostichus halicti and Parasitodiplogaster larvigata. Light microscopy and a video-capture system were used to under-stand muscular movements and reconstruct complicated three-dimensionalmorphology of diplogastrids stoma (Fürst von Lieven and Sudhaus, 2000). Fürst von Lieven (2003) used 96% lactic acid to evert the buccal cavity of Oigolaimella spp., which made it easier to observe the vari-ous structures within it. To confirm the presence/absence and shape of the bursal flap, and vulval and anal morpholo-gy, it is important to observe the ventral aspect of nema-todes. Given these needs, Hooper (1986) developed several methods based upon fixed materials and glycerin-mounted materials. However, some of these methodologies require long times, special chemicals, and/or specific facilities, e.g., TEM and scanning electron microscopy. New simple, quick, and easy methods are needed for routine observa-tion. Here, some methodologies are outlined for the observa-tion of stomatal morphology and ventral aspects of nema-todes using silicone grease or petroleum jelly. The method is as follows. First, put three to four small spots of silicone grease and a drop of water, buffer, or fixa-tive (depending on the nematode species and sample condi-tion) on a glass slide (Fig. 1). Second, pick up and transfer the nematode material(s) to the drop of fluid, put a coverslip on it, and push it gently to adjust the thickness of the fluid layer to the body diameter of the nematode. Third, examine the orientation of the nematode under a dissecting micro-scope and move the coverslip using forceps or a finger to turn the nematode so that it is ventral-side up. The silicone grease holds the coverslip like an agar pad, and the materi-al can be observed using an oil lens without sealing. Silicon grease is most suitable for this observation technique because it is neutral (does not affect pH) and insoluble in water, but petroleum jelly is also available as an alternative to silicon grease. The nematodes can be turned in different directions, and thus, a single individual nematode can be observed from different orientations, e.g., right lateral, left lateral and ventral views can be obtained from a single individual (Figs. 2A-G, 3). If necessary, the nematode can be squashed to evert the mouthparts by pressing the coverslip (Fig. 2H-K). To evert the mouthparts (and pharynx), a spot a quarter to one third of the body length from the anterior, or just behind the cardium, should be pressed quickly. In diplogastrids, the cheilostom sometimes catches on the lip part and gymno-stom, and posteriorly everts from the body (Fig. 2H-K). When examining cultured material contaminated by bacteria (e.g., new cultures established without steriliza-tion), the nematode surface is often covered by bacteria which obscure the observation of surface structures, e.g., genital papillae arrangement and surface annulation and/or striation. Such nematodes can be washed with 0.1-0.5%