worm SEM

can anyone suggest a protocol for SEM of adult worms [that prevents ‘wrinkling’ ]?
the [generic] protocol we follow seems to make them shrink and wrinkle. and they show droplet-like structures on the cuticle [that are artefactual and definitely not part of the worm…not even agar pieces].

Some suggestions to try would be to do a dehydration series with more steps… Another consideration is that to deal with the cuticle during critical point drying, leave the worms in there for very long periods of time for each change of c02. I can’t remember exactly, but the whole process should take a couple of hours (this helped quite a bit with worms that are more delicate than C. elegans.) If you smell any ethanol (or whatever solvent) after critical point drying, do more changes in 100% c02, or do longer changes. Possibly consider sonicating the worms in a water-bath sonicator for maybe 5 minutes prior to fixing them… this will help them be clean. Doing freeze substitution is another thing you could try as a gentler way to dehydrate them… it is good to have a gadget for doing such a thing but it can be done quite well with a -80 freezer and maybe a -40 or -20 freezer.

There is a section in the WormBook - Methods in Cell Biology that goes over fixation techniques for worms.

Athens, OH