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C. elegans were cultured on petri plates as described by Dusenbery ('73). For fixation animals which had just reached the egg laying stage were rinsed from plates of synchronized animals, anesthetized for ten minutes with 1% propylene phenoxetol fixed for one hour at room temperature in 2% OsO4 in 0.1 M cacodylate buffer, pH 7.4, sectioned just posterior to the end of the oesophageal musculature, stained for one hour in 1% uranyl acetate in 0.1 M maleate buffer, pH 5.9, and embedded in epon. A persistent problem due to the toughness of the cuticle has been incomplete penetration of the resin except near the cut end. For microtomy individual heads were cut out of the flat mold and attached with epoxy to supportive blocks. Serial sections were cut on a Sorvall MT2B ultramicrotome at thicknesses appropriate to the complexity of the structures being examined, 120 nm where the axes of nerve fiber bundles being followed for long distances were perpendicular to the plane of the section, 50 nm in the CNS where fibers are circumferential and were therefore cut more nearly parallel to theIr axes. The procedure was systematized so that the cutting rate was regularly 300 sections per hour. Ribbons were collected on slot grids covered with carbon coated formvar and stained five minutes in lead citrate No more than 1 or 2% of the sections were lost. Electron microscopy was carried out using a Zeiss EM9 for which a camera capable of holding a 100-foot roll of 70 mm film was built to expedite the making of the approximately 15,000 low magnification electron micrographs used in this study. All micrographs were made on Rekordak microfilm.
Since the nerve and muscle processes discussed look very much alike except at their terminal specializations and intracelluiar staining techniques are impractical in such a small animal, there is no possibility of linking central with peripheral processes except by extensive three-dimensional reconstruction from serial sections. That is, each process must be followed in its entirety through serial elctron micrographs in order to describe it correctly. For convenience in accomplishing this we used an electron microscope serial section cinematographic procedure similar to that first described by Levinthal and Ware ('72). For this procedure an apparatus, shown in figure 1, was constructed which enables micrographs of sequential sections to be aligned and photographed with a 35 mm movie camera. With this apparatus 35 mm movies of up to 800 micrographs of sequential sections have been created on microfilm at a rate of 60- 80 frames per hour In order to make full use of the inherent resolution and contrast range of the microfilm, movies were used in a Vanguard M35 projector which had been modified so that the film plane could be viewed directly with a room dissecting microscope. All results presented derive from this cinematographic analysis of serial sections.
The work reported has been done primarily on three animals which were sectioned perpendicular to the body axis completely from the snout through the nerve ring. Approximately 1,100 120 nm sections were required to cover this distance in the adult. Shorter series, encompassing only the nerve ring (500 50 nm sections) or only the cephalic end of the animal from the tip to the beginning of the oesophageal musculature (about 200 50 nm sections) were made in five additional cases in order to determine the extent of variability from animal to animal or to resolve difficulties presented by obliquely cut membrane surfaces.
Web adaptation, Thomas Boulin, for Wormatlas, 2002