Worm Breeder's Gazette 8(3): 97
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
In the last few months, several people have asked about the CGC strain freezing procedures. For general reference, here are some detailed notes and observations on freezing and thawing for optimum survival. Two important things. The most important thing is that you have lots of L1's and L2's to freeze, as they are the best survivors. L3's and L4's seem to survive at a lower rate, and adults do not survive well at all. The other important thing is that freshly starved worms seem to survive freezing better than well-fed ones. Getting lots of L1's and L2's to freeze. Homozygous stocks are generally pretty easy. If you start a large agar plate (15 x 90 mm, streaked with a grid of OP50) with ten L4 hermaphrodites, the plate will starve when the F2's have recently hatched. This is a very happy thing, and you can proceed to the actual freezing. With heterozygous stocks, you'll need to start a few clones on small (15 x 60 mm) plates, check the progeny for correct marker segregation, and pick three confirmed clones. When these starve, wash off all three plates and pool the worms for freezing. Other types of stocks are a little more tedious. These include burrowers, male stocks, paralyzed or severe Dpy and other slow-growers. With any of these types, it sometimes helps to start three or four small plates with one to five L4's, or three or four cross plates in the case of male stocks. As soon as you get a bunch of L1's and L2's on the plates, wash them off thoroughly, pool and wash the worms free of bacteria by centrifugation and removal of the supernatant. Resuspend the worms in 2 ml of M9 buffer and put them on a shaker at an appropriate temperature for 12 hours or so. Overnight is fine. This starves them. Before freezing, add one drop of an overnight OP50 culture so the worms will have plenty to eat when thawed, and freeze as usual. This isn't as much work as it sounds, and gives you a warm feeling later. It is especially useful with burrowers (most wild-type C. elegans isolates), of which there are usually none left on the surface when the plate starves. Actual freezing. Having obtained lots of larvae in a starved state, this is what I do: (1) Wash larvae off the plate with 2.2 ml of M9 buffer, draw the buffer/worm suspension into the pipette, measure ( usually 1.6 to 1.8 ml) and place the suspension in a sterile 30-40 ml test tube with closure; (2) add an equal volume of S medium plus glycerol (this is 30% glycerol, w/v, so the final is 15%); (3) vortex gently to mix; (4) draw up 3 ml in a sterile 5 ml pipette; and (5) dispense in 0.6 ml aliquots to five 2 ml Nunc freezer vials. These vials are placed in holes drilled into a styrofoam block which has been fitted into a styrofoam box that has a lid (Lew and Miwa, WBG 4: #1), and the whole thing is put in a -80 C freezer. Leave them there at least three hours; I usually leave them overnight. Then you can put them in liquid nitrogen. Four of the vials go into twin LN2 refrigerators; the fifth is put in liquid nitrogen in a separate place to be thawed the next day as a survival tester. Survival and contamination checking is done on the third day after thawing. If you have three small plates instead of one large, start with 2.5 ml of buffer to allow for what soaks into the agar. Wash one plate and draw the mixture back up, wash the next two plates with that. If you're doing the shaker trick, just add an equal volume of S + glycerol, vortex and dispense. It helps to use disposable graduated conical tubes with screw caps, since you can easily see how much S + glycerol to use. You can also prepare a single freezing solution, a 50/50 mixture of M9 buffer and S + glycerol, and use that to wash off the plates. You can either start washing with enough solution to leave 3 ml for dispensing into vials, or wash with a smaller volume and bring it up to 3 ml just before dispensing. Thawing. In my experience this is not terribly critical, but I try to do it the same way always. Vials are removed from liquid nitrogen and placed on the lab bench, and a timer is set for ten minutes. When the timer goes off, you'll usually have a chunk of ice with a layer of liquid around it in the vial. If it's still frozen solid, rub it briskly in your hands until the pellet starts to melt. Wipe the vial dry with a Kimwipe. This keeps condensed water from the outside of the vial from falling onto the petri plate. Vortex with the cap still on to mix the stuff into a runny slush and dump it into a clean, labeled agar plate. Results. At the beginning of the first Genetics Center contract, N2 freezing survival testers were frozen to provide monthly and yearly tests over the entire contract period. Survival rates from those testers averaged 26% with no decrease in survival in stocks frozen for the entire 5-year period; fluctuations around that average are presumably due to sampling variations. Long-term survival tests will continue. Survival of stocks stored at -80 C in a Revco is as good as in liquid nitrogen over an initial 6-month test period.