Techniques for Preparing Crustaceans for Scanning Electron Microscopy

Several techniques for preparing the internal and external surfaces of crustaceans for SEM are described in detailed flow-chart form. Suggestions are given for improving fixation, cleaning, and overall appearance of specimens. The study of gross morphological detail as well as fine structures of crustaceans has been substantially enhanced by the many recent technological advances in scanning electron microscopes (SEM), and, as a result, the use of this instrument in all aspects of crustacean biology is on the increase. No matter how good an instrument may be, however, the results still depend on the quality of the material photographed. One of the major problems in preparing crustaceans for SEM is the removal of accumulated debris. An additional problem is that the crustacean cuticle provides an excellent habitat for epibiontic organisms ranging from bacteria and fungi to symbiotic mites (Bauer, 1975; Felgenhauer and Schram, 1978; Holmquist, 1985, and references therein). Apart from aesthetic considerations, these organisms may cover and obscure important structures, such as the terminal pores of chemoreceptors, or cause difficulties in the identification of setal types. Marine crustaceans are particularly troublesome, since they may exude appreciable quantities of mucus at the time of fixation. Several authors have offered good suggestions for specific problems in preparing crustaceans for SEM (e.g., Abele, 1971, gonopods of brachyuran crabs; Scotto, 1980, larval crustaceans). This note describes several general techniques that have proven to be particularly successful in my own work (e.g., Felgenhauer and Abele, 1983, 1985) for freeing the body surfaces of crustaceans of unwanted organisms and debris. Several other methods are presented for examining the internal anatomy of crustaceans. All micrographs presented in this note were taken with a Cambridge S4-10 or a JEOL 840 scanning electron microscope at accelerating voltages of 3-20 kV. I. BASIC SPECIMEN PREPARATION Fresh material is always preferable to specimens previously fixed in ethanol or Formalin. Below is an outline of a simple protocol that consistently produces excellent results (Fig. 1A, B). la. Fix fresh material in 3% glutaraldehyde at room temperature for 3 h in whatever buffer is best for the tissue being prepared. For fresh-water forms, I prefer a 0.1 M phosphate buffer at a pH of 7.0, and for marine crustaceans I use Millipore-filtered sea water or 0.1 M sodium cacodylate at a pH of 8.0. 1b. If Formalinor ethanol-fixed tissue must be examined, hydrate it to distilled water and continue beginning at step 3 below. 2. Wash tissue in 3 changes of the chosen buffer for 5 min each to remove excess fixative. 3. Accomplish secondary fixation (postfixation) of the material in 1-2% osmium tetroxide (OsO4) in buffer choice for 2 h. Always use OsO4 under a