Worm anesthesia

Model Organism: Worms Methods & Reagents
1

Hi,

We are trying to follow developmental processes in L3 and L4 worms by microscopy for up to 8 hours. We need to keep the worms as quiet as possible but obviously also without affecting the processes we want to study.

We are currently trying combinations of levamisole and tricaine but the worms are irreversibly affected after long exposures. Can anyone help with tricks/experiences/references etc?

Thanks in advance!

Peter

2

The use of levamisole is described in various WormMethods in WormBook. These may be of help.

http://www.wormbook.org/db/misc/search.cgi?search_html=on;search_preprints=on;query=levamisole

3

Hello,

have you tried to phenoxy-propanol (C. elegans I book)? Or the method John Sulston used for lineageing?

Also, what about using non-toxic glue, Bill Shafer glues his worms down to do the life recordings of chameleon in the pharynx and in neurons.
That works. you might just have to try to glue the worms in such a way as to not obscure the area you are interested in.

Good luck.
PS: if you get interesting replies by email, please be so kind as to post a summary back to the board. Thanks.

4

I tried 2-phenoxypropanol when observing worms with GFP-tagged neurons and found that it dissolved the neurons.

5

I admit that I have never tried this for long periods, but for short term anaesthesia you can calm the worms by adding ethanol to M9 medium. I believe they will eventually adapt and reawake, but you might play with 5% or 8% ethanol to quiet them briefly. Maybe in combination with the superglue trick you can fasten them to a substrate (to maintain a standard orientation), and add ethanol just before each observation to quiet their body motion long enough to obtain new images?

For short term experiments, we have often used 8% ethanol to make worms straighter just prior to fixation. We see no defects in anatomy at the EM level caused by this exposure. Stronger agents like sodium azide are expected to cause damage. Even when under anesthesia, when prodded with a razor blade, male worms are more prone to sudden curling than hermaphrodites.

David Hall

6

You mentioned that the worms were affected by long exposures, light particularly of high intensity, is actually toxic to the worm. Is it possible to limit light exposure in your experiments?

7

Has anyone tried CO2?

8
Has anyone tried CO2?
I've seen a high-power dissecting scope on which someone had rigged a tube coming from a CO2 tank (with, obviously, a regulator, etcetera) so they could sent a jet of CO2 gas onto the center of a plate to immobilize worms while they looked at them, and they reported some success (using the CO2 they could readily examine fine structures using a GFP reporter, structures that they couldn't otherwise examine easily because the animals tended to move too much); but I never used the setup myself, so I don't know how well it worked, I don't know for how long the worms could be immobilized (the person using the setup used it for brief periods, not for anything like 6-8 hour period the original poster in this thread envisions), I don't know how long the animals could be immobilized and recover well, and I don't know whether the lethargy-inducing effect was because of high CO2 concentration or low temperature (or whether either effect of the gas, especially the temperature, would cause problems with the underlying biology if the idea was to immobilize a worm and watch it develop/respond to stimuli/etcetera).