Protocol for "Cryopreservation of dehydrated Caenorhabditis elegans with multiple recoveries using a granular medium"

Reagents, Materials, and equipment

1. S buffer (Stiernagle 2006)
2. Glycerol (e.g. Sigma G5516)
3. 15 mL conical centrifuge tube (1 per strain)
4. 1.8 mL cryogenic tube (1 per strain)
5. 6 cm diameter NGM OP50 plates (Stiernagle 2006) (2-3 per strain)
6. Platinum wire pick
7. Sterile transfer pipette
8. Airtight box (approximately 28 X 19 X 8 cm, e.g. Rubbermaid 1991158)
9. Rack for cryotubes (must fit into the airtight box)
10. Desiccant (anhydrous calcium sulfate with cobalt chloride indicator, 8 mesh size, Drierite, W.A. Hammond, 20 g)
11. Cryostorage box
12. Stainless steel spatula
13. P200 and P1000 micropipettes
14. Centrifuge
15. Microbalance
16. Vortexer
17. Stereo microscope
18. Cornmeal (e.g. Bob’s Red Mill Medium Grind, #1148S24), 0.6 g per tube

Cornmeal preparation

1. To sterilize cornmeal, place on a piece of foil and heat in an oven or hot plate for about 10 minutes at 120-150°C.  Do not let the cornmeal turn brown.
2. Transfer cornmeal to a sterile, airtight container for storage.
3. Note: when handling dry cornmeal, use a metal rather than plastic spatula to reduce effects of static electricity 

Dehydration and freezing procedure

1. Pick five gravid adults onto each of 2-3 standard 6 cm OP50 plates and incubate at 20 C for three weeks. 
2. Note: Incubation can be reduced to 1 or 2 weeks with reduced survival rates
3. Plates should be starved and some dauer larvae should be present. 
4. Prepare a stock solution of 30% w/v glycerol in S buffer 
5. Use a sterile transfer pipette (see Note 1) and several mL of S buffer to wash the worms into a 15 mL conical centrifuge tube. Add S buffer to a final volume of 10 mL. 
6. Pellet worms by centrifuging for 5 minutes at 700 rcf. Use a sterile transfer pipette to remove the supernatant. Add fresh S buffer to 10 mL, pellet worms, and remove supernatant again, leaving < 100 µL of S buffer with the pelleted worms. 
7. Use a sterile transfer pipette to transfer the pellet to a 1.5 mL microcentrifuge tube. Add S buffer to 125 µL using a microbalance to aid with measurement (see Note 2).
8. Add 125 µL of 30% glycerol in S buffer and mix by gentle vortexing or flicking the tube. This is the “worm mixture”.
9. Add 0.6 g cornmeal to a 1.8 mL cryotube. 
10. Pipette 200 µL worm mixture into the prepared cryotube. Mix with the pipette tip. 
11. Place the uncapped cryotube upright into a tube rack or other support inside the drying box.
12. Scatter 20 g fresh desiccant onto the bottom of the drying box. 
13. Seal the lid and leave the drying box at RT for 48 hours (see note 3). 
14. Open the drying box. Cap the tube and place it in a cryostorage box at the desired ultracold storage temperature. 

Recovery procedure

1. Remove the cryotube from the ultracold storage. 
2. Use a metal spatula to transfer a small amount of cornmeal/worm grains onto a seeded NGM plate.  Media can be weighed if desired.
3. Return the tube to ultracold storage. 
4. Check for survivors the following day.

Notes:

1. The same sterile pipette can be reused in steps 4-6 provided it is kept in its sterile wrapper when not in use and only used for one strain. 
2. A microbalance can help with this step. Weigh and tare the empty 1.5 mL microcentrifuge tube and assume an approximate density of 1 g/ml for the S buffer worm mixture. 
3. Using a sealed box and the ratio suggested, we observed little or no color change in the desiccant. Excessive color change could indicate that the drying box is not well sealed. To recharge desiccant, follow the manufacturer’s instructions.

References

Hayden, J., Fang-Yen, C., 2021. Cryopreservation of dehydrated Caenorhabditis elegans with multiple recoveries using a granular medium. microPublication Biology. 

https://www.micropublication.org/journals/biology/micropub-biology-000488/

McClanahan, P.D., McCloskey, M. Ng Tung Hing, D.M. Raizen, C. Fang-Yen, 2020, Dehydrated Caenorhabditis elegans stocks are resistant to multiple freeze-thaw cycles. G3. https://www.g3journal.org/content/10/12/4505

Stiernagle, T., 2006, Maintenance of C. elegans, Wormbook, ed. The C. elegans Research community, WormBook, doi/10.1895/wormbook.1.101.1, http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html

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