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.

Longitudinal Sectioning of Complete Worms for Electron Microscopy after Laser-Induced Fixation

Udo Mnzner and Einhard Schierenberg

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.