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Avery L (1985). freezing worms in soft agar. Worm Breeder's Gazette, 9(1), 88. Unpublished information; cite only with author permission.
Worms must be frozen slowly, if a large proportion of them are to survive. Unfortunately, they sink to the bottom of the tube while freezing, so that the entire tube must be thawed to get them out. Thus, depending on the compromise between freezer space and strain security chosen, a strain must be regrown and refrozen every few times it is thawed, exposing it to hazards of spontaneous mutation, confusion with other strains, or outright loss. I have been freezing worms in 0.2% agar. The agar supports them while they freeze, so that one need only scrape out about 0.1 ml of ice with a spatula to thaw them. The agar seems not to affect survival, but I have only a few months experience, and the sickest strains I've checked have been CB190=unc-54(e190), and CB128 = dpy-10( e128). (The latter is said to be difficult to freeze.) Protocols are identical to Mark Edgley's (WBG 8(3): 9798) up to the actual freezing step. Then I cool the worms suspended in liquid to 0 C, add an equal volume of S medium + 30% glycerol + 0.4% agar, melted and cooled to 50 C beforehand, vortex, aliquot (0.2% agar is soggy enough to pipette), and freeze in styrofoam blocks as usual. To thaw some worms, I sterilize a pointed spatula with flaming ethanol, scrape out about 0.1 ml of ice with it, and dump the ice in the middle of a 6 cm NGM plate spread with OP50. I put 1.8 ml in a tube rather than the 0.6 ml that Mark uses, so that each is good for 10-20 thaws. Naturally, this trick is most useful when one has many L1's and L2's to freeze, since only a small volume of ice need be thawed to get some survivors. I grow my worms on 10 cm enriched plates, which contain 0. 5% peptone (double that in NGM) and 0.1% yeast extract, in addition to the usual ingredients of NGM. The extra goodies support a dense bacterial lawn, and therefore a large crop of worms. I also use 3% agar in these plates (rather than the usual 1.7%), which somewhat reduces burrowing.
