Scanning Electron Microscopy of Trematodes Embedded for Transmission Electron Microscopy

Abstract

Chipping plastic from the surface of trematodes embedded for transmission electron microscopy permits high resolution scanning electron microscopy of surface features. The surfaces of the trematodes Megalodiscus temperatus, Diclidophora merlangi, and Schistosoma mansoni are described and comparisons are made with previous studies using the transmission electron microscope. The structure of the integument of Schistosoma mansoni as visualized with the scanning electron microscope (SEM) has been reported in at least six publications to date (see Miller et al., 1972, for review). Occasional isolated scanning micrographs of other trematode species have also been published but in no instance has the information obtained been limited by instrument performance. Instead, results have been limited by methods of specimen preparation, usually involving fixation, dehydration, and air drying with a subsequent distortion of surface morphology. Critical-point drying techniques should improve specimen morphology although this technique has seen little use in studies of platyhelminth morphology. The purpose of this paper is to describe the results of examination with the SEM of fractured, plastic-embedded trematodes by the use of a technique recently described by Humphreys et al. (1973) as fractography. This technique makes it possible to utilize fully the resolution as well as depth of focus of the SEM in the examination of specimens prepared by routine methods for transmission electron microscopy. MATERIALS AND METHODS Specimens of Megalodiscus temperatus, Diclidophora merlangi, and Schistosoma mansoni which had been previously fixed, dehydrated, and embedded for transmission electron microscopy were used in this study. Any method of fixation which is satisfactory for transmission microscopy can be used. The embedding media used include Epon, Epon-Araldite mixture, and Maraglas. The blocks were mounted in vise-type holders and the plastic embedding medium removed with sharp razor blades. Excess plastic was first trimmed off and then the razor blade was sliced into the plastic close to, and parallel with, the specimen Received for publication 26 April 1973. surface. An outward flick of the razor blade caused substantial portions of plastic to separate from the worm surface. Entire worms could, in some instances, be separated from the surrounding embedding medium. The fractured specimens were oriented on specimen stubs and attached to the surface with a small drop of synthetic mounting medium. The specimens were then coated in a Denton DV-502 vacuum evaporator with a thin layer of gold-palladium and examined with an AMR-900 SEM. RESULTS AND DISCUSSION Low-magnification micrographs of the surface of Megalodiscus temperatus show the undulating surface to be covered with numerous, small protrusions (Fig. 1). Figure 2 shows the same surface at high magnification and demonstrates the surface detail which is visible in tissue prepared by this method. Although the spherical surface protrusions are seen in some transmission electron micrographs (see Bogitsh, 1968), their true contribution to the surface morphology of this worm is apparent only in high resolution scanning micrographs. Low-magnification scanning micrographs of the anterior dorsal surface of Diclidophora merlangi reveal not only the folded surface previously described by Morris and Halton (1971) but also numerous parallel rows of domelike surface protrusions, each of which has a diameter of about 6 to 8 /i (Figs. 3, 4). The microvilluslike structures described by Morris and Halton (1971) are numerous over the lateral surfaces of the protrusions and the intervening regions, but are absent from the apices (Fig. 5). In many instances the tips of the microvilli are broken off, leaving only small stubs behind. Figure 6 shows a low-magnification scanning micrograph of a partially fractured block containing male and female specimens of