Worm Breeder's Gazette 11(4): 117
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
In order to follow the extension of cellular structures along the anterior-posterior axis it would be desirable to make longitudinal sections through complete worms. Sectioning a worm longitudinally is certainly more economic than cutting transverse (needs only about 10% of sections). Three dimensional reconstruction of cells and tissues is probably easier in many cases with longitudinal sections. Therefore, we have developed a method for doing this using a laser microbeam as a tool for careful fixation. Young adults are transferred from an agar plate into a drop of 2.5% Glutardialdehyde in 0.02M Phosphate buffer with 50 l/ml of a saturated solution of Trypane Blue. Worms are slightly squeezed in this solution between a microscope slide and a coverslip to make sure that they are perfectly flat. With single pulses of a laser-microbeam ( wavelength 550 nm, Rhodamin 6G) coupled to a microscope, tiny lesions are made into the cuticle. The thin layer of blue dye allows absorption of the orange laser beam. We had found earlier that other fixation methods for whole worms not involving multiple penetration sites along the body gave only satisfactory results in the anterior part of the animals. A hot aldehyde-peroxide method described for C. elegans by Byard et al., 1986 (Stain Tech. 61, 33) did not work well in our hands. Therefore we punched tiny holes with the laser into the cuticle starting from posterior to anterior. On each side 6-10 lesion are made waiting 30-60 sec after each pulse to allow penetration of the fixative. After treatment fixation in Glutaraldehyde is continued for 2 hours. Animals are then embedded individually into small agar blocks, washed in PBS (similar osmolarity as Glutaraldehyde + Trypane Blue) and postfixed in 2% OSO4 for another 2 hours. Later they are embedded in Araldite. With this method we are able to make perfect longitudinal sections through young adults from tip to tail. Fixation is of equal quality along the body. Besides holes in the cuticle the laser-induced lesions cause only tiny damages in the hypodermal and muscle layer, if at all. As the fixative does not penetrate the eggshell, so far we have chosen animals which do not carry fertilized eggs. For laser- induced fixation of eggs, see Cole & Schierenberg, 1986 (Experientia 42, 1046). With the help of longitudinal sections we can well study ultrastructure and extension of tissues like the alimentary tract, body muscles or the gonad including the distal tip cell. We imagine that this method may also be helpful for the analysis of other structures like the nervous system.