Recently we tried to revive our N2 worm stock (stored at -80C, prepared in 2014).
When the stock is thawed, we put everything on the NGM plate.
After few minutes, the worms started to twitch and move a little, thus we incubated them at 16C and 25C separately (we thawed on few plates).
However, after few days, the worms were dead. We did notice traces of worms’ trail on the agar, meaning the worms did revive.
One thing we noticed is that although the worms were crawling, they crawled around the same spot where they were first spotted.
I read somewhere that glycerol is somehow toxic to the worms, so it might cause death to the worms as they ate the bacteria stained with glycerol?
But at the same time, we revived another mutant strain using the same technique, and the worms able to survive and propagate eventually.
The mutant strain stock is newer, prepared in 2016. Will the age of stock has some effect on recovery?
Any ideas or tips on successful worm recovery?
Thank you for your time and have a nice time:)
Were the animals you froze starved L1s? The way you talk about them makes it sound like they were older, and larger.
- Whenever you freeze strains, you should wait a day or so and do a test-thaw to make sure it went OK, even if you don’t expect any to fail. The easiest way to do this is to freeze an extra tube, and thaw it when you transfer the other tubes from the styrofoam box in which you freeze them to a longer-term storage space.
- Given reliable freezers (or rapid responses to freezer failures), a good frozen stock should remain highly viable for a very long time - certainly for many years, and usually for a couple of decades.
In my experience, the surviving rate definitely decreases with the age of stock. But drying the plate as soon as possible appears helpful to worm revival. Thus I always try to suck up the worm pellet (worms usually fall into the bottom during freezing) to a new NGM plate instead of putting everything on the plate because too much water may choke the worms. I also let the plate open for sometime (e.g. several hours or overnight) to dry up the plate. Hope this helps.
Increasing the volume of freezing solution is also good for worm recovery, as reported by the Mori lab (http://www.wormbook.org/wli/wbg17.2p22/).
We froze L1 starved animals, right after they hatched. Thank you for your information
Maybe I can try this. I took up all the liquid and dispense them in a few drops on the agar.
Will try to take the bottom one only. Thank you for your suggestions
We can try this during frozen stock preparation next time. Hope it will help in the future. Thank you for your suggestions.
Just thinking, will it help if I drop the worms somewhere at the plate without the E. coli (Maybe I can place each at a side, and the worm will crawl towards the food)?
Previously I dropped it on the E.coli, so when they revived, is it possible they died of glycerol toxicity after consumed the stained food?
We do try and place the worms from the freezer on the side of a plate with a spot of food.
This encourages any healthy worms to leave the freezing medium and crawl into the food.
This makes them easier to find, if there is a low recovery rate (but it doesn’t work for severe Uncs).
Also, I believe you should use L1s that have been starved for several hours (rather than immediately after hatching).
At least the stock you could not recover was N2, not a precious unique strain!
Good luck, Janet
I would agree with Janet Duerr about not using freshly hatched L1s - wait until the mothers have stopped laying eggs and all the eggs have hatched. Obviously it will help if your strains are free of bacterial or fungal contamination (some bacterial contaminants don’t get as easily cleared as E. coli and so the animals don’t starve as well, contaminants can soften the agar so the worms burrow and greatly decrease the yield from washing off the plate, and even when fairly harmless mold can get in the way).
I don’t have an opinion about limiting volume put on the plate, or about putting the worms on the food or off it, but suspect it does help if the plate can rapidly absorb the liquid placed on it - a somewhat dry large (10 cm) plate or a rather dry smaller (6 cm) plate is a good idea.
Janet Duerr and HillelSchwartz, thank you so much for the valuable insight.
We shall try out and see what will happen
Just to make things clearer, which storage method is better?
- Let the gravid worms lay egg and progenies hatch (at this point the food is usually depleted in our case, but maybe there are still traces of food and might not starve enough?)
- Bleach the gravid worms and place the eggs on fresh NGM without food, until the worms (L1) hatch
Appreciate opinions on this matter, thank you
- I always let the freezing plates starve at least 24 hours prior to washing the worms off and into Freeze solution. The exception is a daf-16 mutant, which you need to catch when very recently starved, i.e. <24 hours (Otherwise, recovery is poor.)
- I freeze by putting cryotubes into styrofoam blocks, bottom and lid: a slow freeze permits better survival than a quick, hard freeze. I later unpack the styrofoam blocks and file the tubes into the strain collection. For styrofoam blocks, I just re-use the blocks our disposable 15 ml conical tubes come in.
- I always test thaw after a few days. Typically, most strains recover great. As Hillel noted, bacterial contaminants can compromise that, as can a daf-16 mutant if not properly frozen.
- I test thaw every strain onto seeded and reasonably dry large plates (10 cm). And I flick the tube a few times to suspend any settled worms, though as Janet and Hillel note, it is L1s that recover, not the big ones.
Same for normal thaws. You can also aliquot your thaw over four small plates. Why? The worms do not like glycerol, and it can compromise recovery. So be careful with those large volume freezes: you may have too much glycerol when you thaw.
I would not bleach and hatch off. To my knowledge, bleach is an unknown variable for freezing. And why bother? Just starve out a few plates and wash the worms off. And yes, make sure they are starved. There is evidence that it is L1 arrest, presumably an energetic arrest of sorts, that promotes good survival of freezing. And that is why the daf-16 mutant survives poorly if starved for too long.
This is very helpful. Thank you