Can anyone suggest me a best protocol for freezing worms using glycerol storage? I've also tried the protocol from wormbase and faced more trouble shoot in reviving the worms though it has been stored with sync population during the starved condition. Can anyone help me out in this regard?
I think most of the written freezing protocols are more or less the same. Make sure the following points.
1)glycerol concentration. carelessly measured glycerol changes the final concentration.
2)worms need to be L1, but not dauer. Freshly starved plate is good but not starved several days
3)freeze slowly. Use styrofoam box or cell freezing device to slowly freeze the mixture
4)pour the worm right after thawing, leave it longer seemed decreases the survival of animals.
Dear Tokiko Furuta , at what concentration/ percentage that glycerol and worm's ratio should be freezed?
I used freshly synchronized L1s (bleach to kill everything except eggs), put eggs on "unseeded" plates (no bacteria), and then washed & froze.
Or you can grow up a larger population & watch for eggs 1000s-10,000s. Check every few hours for L1s, then wash & freeze.
I think we did about equal volumes of worms & glycerol, but the protocol should say for sure.
Remember, only the newly-hatched L1s can survive the freezing process. Everything else dies.
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