I had a quick question about storage of my worms. I am looking for a method alternative to freezing
with liquid Nitrogen or having a -80 degree Freezer. Lab space and equipment are very limited.
any suggestions?
I’m not aware of a good alternative. From everything I’ve heard, you cannot store worms at -20 (I’m not sure exactly where the cutoff is, or even if anyone has done the experiment; stocks that warmed to -60 for a time have been fine, but as there’s no distinction between a -60 freezer and a -80 freezer this is moot).
In theory, you could maintain stocks of arrested worms or especially dauers at 10 degrees; certainly this is often a valid strategy for months at a time, even up to 6 months or so. But it’s never a reliable strategy (some cultures make it that long, others don’t, likely for stochastic reasons of airtight seals forming and causing anoxia, or of contamination, or dehydration, or who knows what else), and even if it were to be completely reliable for a time (say 4 months, with redundant copies of each plate) it would require significant labor to maintain, on an ongoing basis, and even more unlikely organizational capacity; I would fairly confidently predict a system of maintaining such stocks would break down within a year or two.
For some other, non-Caenorhabditis species it’s straightforward to isolate large numbers of dauers or dauer-like infective juveniles, which can then be maintained in water in cell-culture flasks for a year or more - but, again, this approach has significant reliability and organizational issues, and I don’t think it’s remotely as easy to collect and store dauers in Caenorhabditis.
I think you have to bite the bullet and invest some money (but not that much space) in buying a -80 freezer or a liquid nitrogen flask. We chose the former as it was more convenient for us, the price was also OK. If you have on-site deliveries of liquid nitrogen (AND SOMEONE TO LOOK AFTER IT) then that might be a better option.
If you are in a lab or department where there are others undertaking cell/molecular biology research, then perhaps you could beg a bit of space in their freezer or dewar flask???
Steve
appreciate the feedback. what about methods for slow freezing? to get worms at the level where I can eventually get them into the nitrogen tank?
The simplest method for slow freezing is to take two styrofoam racks for 15 ml conical tubes, put the cryotubes containing your worms in your freezing mixture in one rack, invert the second rack and use it as a lid (the cryotubes are somewhat taller than the depth of the holes in the rack, so you need to line up the holes in the second rack). Hold the two racks together fairly tightly with a rubber band or two, and stick in the -80 freezer for a day or more. After that time, transfer the tubes to standard freezer boxes (far, far more space-efficient than the bulky styrofoam package) or to liquid nitrogen, and do a test thaw of (or, if you used agar, from) one tube (for each strain frozen).
Hillel,
We don’t make any special effort to slow freeze worms and we haven’t encountered any strains where survival after thawing is a problem. Do you know of specific cases where
slow freezing is key?
Happy New Year!
Eric
When I learned to freeze worms (I no longer remember whether someone showed me or I read a protocol), this is how I learned to do it, and I’ve never tried freezing elegans without insulating them in this fashion. This is I think the standard method (see for example Theresa Stiernagle in WormBook) - but I don’t know who if anyone ever demonstrated that it actually makes a difference. It would certainly be convenient if it turned out to be a waste of time and space to freeze the worms in styrofoam before transferring them (admittedly, a small waste of time and space) - but given force of habit and received wisdom I’d have to see the data from someone who bothered to do a head-to-head comparison with and without styrofoam before I stopped using it.
yeah, slow freeze is the standard protocol and not much trouble to do. i did do the comparison one time with N2:
slow (styro box in -80), vs faster (on metal shelf in -80) vs really fast (straight into liquid N2). on thawing they
all had pretty good survival. some genotypes can be finicky, so maybe it makes a difference for them. the ones
i have had trouble with didn’t seem to do much better with slow freeze (admittedly anecdotal).
May i know how long could a cryovial last? For many years or we will have to re-stock them from time to time?
Because long before we managed to get a freezer, i once stored our frozen worm stock (quick freezing method into liquid N2)
and stored at -80C freezer at neighbour lab for 2 years. Recently, i found that the recovery was very low or even zero recovery - i was suspecting that if it was due to temperature shift (because i moved from liquid N2 to -80C) or it could be the freezer issue?
If you have reason to think you made a good frozen stock (ie you made several identical vials when you froze them, and did a test thaw of one vial a few days after freezing them, and the test thaw was good), then the frozen stock should be good for many years at -80 and for decades in LN2. I have heard it claimed that the freezing protocol without agar is better for long-term storage.
I’m not familiar with your “quick freezing method into liquid N2” for elegans; did you do test thaws? And if your neighbor has a -80 freezer, couldn’t you borrow the space in that freezer for a couple of days so that you could freeze your worms according to the standard protocol?
what i did was, i snap-frozen the glycerol stock back at that naive time (in contrast to the slow-freezing in standard protocol)…
anyway, now that we have our own freezer, so i will just carry out the standard protocol 
Thanks for the input
“Book 'em, Danno.”
we have the motive (no freezer available) and the murder weapon (snap freezing)
Case closed.