M9 wash buffer

My partner and I are highschooler’s embarking on our first C. elegans research questions and we had a few basic questions. For one, we want to treat worm with a toxicant by keeping them in a microcentrifuge tube suspended in our M9 buffer/toxicant. Will keeping them suspended in M9 in a tube for some time, up to 24 hours, alone kill the worms? Will a lack of oxygen kill the worms?

For a standard synchronization worms are also left overnight in M9, so that shouldn’t be an issue. I usually do it in a 15 mL tube with 10 mL M9 and place it in a rotator, that provides plenty of oxygen. I guess 1 mL in a 1.5 mL tube should be fine too.

However, if there is no food in the tubes, they will go into L1 diapause, so you won’t be looking at normally developed worms. What do you intend to do after the 24 hours?

We just want to suspend the worms in a mixture of M9 and a toxicant. We didn’t want to mix the chemical into the agar or pipette it onto the plates. Realistically, we would only be leaving the worms in the tubes (we are not sure what life stage yet) for AT MOST 30 minutes, more likely 15 for a really acute exposure. If we were using L1s, I’m assuming this would not result in arrest, and if we were using L2/L3/L4, would it also be fine?

just 30mins of incubation with M9, should be good for any stage of worms. just have appropriate controls. good luck.

Right, I didn’t think about the fact that you would likely be using adults (or later larval stages). In that case 30 min should be fine, but I definitely wouldn’t leave them much longer than that. If you do at a later stage want to test a more chronic exposure, you can always use a liquid culture with bacteria, but that’s more work. Good luck with the experiment!