Freezing of C. elegans to super low temperatures


I am curious about why and how freezing to -80C in C. elegans works. I’ve found some references on the general process, specifically the involvement of IIS and trehalose, and general nematode strategies for cold tolerance, but could not find any information on pathway specifics. What is known about why and how C. elegans can be frozen to ultra low temps and how they “come back” out of this? It’s gotta be more than IIS and trehalose because Drosophila have both, but cannot be frozen to ultra low temperatures. In fact, it appears that C. elegans is the most evolved multicellular organism that can go so low and come back “to life?” Any references on the pathways and/or how this process was originally discovered and described would be appreciated.


The method is originally described in Brenner, 1974 in Genetics and hasn’t changed much except for the possible use of soft agar that can be added to the mix that some labs are using.

I have recently done some work on a different type of method to preserve worms but it is not published. A collaborator I have talked with is planning to try that method in flies.

For now there is always this method for fly preservation:

Thank you, Richard. Yes, we’ve used the originally described freezing method many times. I was just curious about its biological underpinnings. What makes the L2 and L3 stages more prone to successful freezing? And what are the pathways that govern that and allow them to successfully resuscitate after thawing? I did not realize that you can freeze Drosophila. A recent quick search suggested that it is not possible, so thank you for the reference. I also found this: which I found to be really fascinating - flies were made freeze tolerant (although to a much higher temperature). Anyway, it is not an area that we actively study, but I find it fun to think about, especially since freeze tolerance is one of the characteristics that makes C. elegans such a powerful model. Going to look for a multi-species review on cold tolerance. If anyone knows of any good ones, please let me know.

I can’t help you with your question, but you might find it interesting to compare with methods used for nematodes other than Caenorhabditis; for example, protocols for freezing entomopathogens involve freezing IJs, rather than L1s as in the elegans protocol.

Since it seems that you’re interested in pathways that promote cryobiosis, it might be worth poking around the tardigrade literature to get a sense as to what the adaptations are present in an organism that can survive extremes of cold, desiccation, radiation, etc.


I always thought that in the original freezing paper they had developed the method simply by trying freezing conditions commonly used to freeze bacteria, and that it just seemed to work.

Thank you all for the info. I guess I was just surprised that not more is known about the pathways that are responsible. Hopefully, someone out there is working on that :slight_smile: Here are some references I found others may find interesting: 1) p. 189 of 2) 3)

It was my understanding that it is the L1s that survive best, due to elevated DAF-16 activity. L2s and L3s survive, but fertility is frequently compromised in those animals. Maybe the L1 diapause protects the germline, since it doesn’t proliferate? Just guessing here.

Yeah, I’m fairly sure developmental arrest is a key part of surviving freezing, especially by comparing to other nematodes whose larvae may not arrest if they hatch in the absence of food.