Pick worms to six 6 cm plates for each strain you wish to freeze.
- For most strains, 4–6 plates is enough. For sick strains, more plates give a higher titre and therefore a better thaw rate.
- Check the genotype of the strain so you can follow any phenotypes and confirm the animals are correct.
Store the plates at the optimal temperature for your strain — 20°C for most — until the plates are starved. The strain is ready to freeze when there is no bacteria and few or no embryos remaining. Most of the animals should be L1 or L2.
Prepare cryovial tubes (Room 223, Shelf 2) with cryo-safe labels printed on the label maker, carrying the strain name and date frozen. Use the "Cryovials Label Template".
- We currently freeze six cryovial tubes per strain: four for working stocks, one for long-term liquid nitrogen storage, and one to use as a test thaw.
- For transgenic strains the lab creates, freeze five tubes per independent line: three for working stocks in the -80°C, one for liquid nitrogen storage, and one for a test thaw.
- When the last tube is thawed, you must re-freeze the strain.
Get an ice bucket.
Heat the freezing buffer (Peggy's recipe) in the microwave very slowly, checking every 30 seconds to see if it has melted. Put it in a 50°C water bath until ready to use.
Loosen the lids of all the cryovials.
With a serological pipette, take about 6.5 mL of M9 for six 6 cm plates and dispense onto the first plate. Wash the worms off, then using the same pipette continue washing worms off all the plates.
Put 1 mL of worms and M9 solution in each cryovial. Less volume can be added to the test thaw cryovial, but remember how much was added for the freezing buffer step.
Put the cryovials with worms on ice for 5 minutes.
Start a timer to track elapsed time of worms on ice.
Add an equal amount of freezing buffer as M9 buffer per tube — 1 mL, except possibly for the test thaw tube.
Vortex the cryovial.
Put the cryovials back on ice for 5 minutes. The elapsed time for adding the freezing buffer and vortexing should be about 5–6 minutes.
Place tubes in a 1.5 mL tube styrofoam container in the -80°C.
Styrofoam rack
Cover the styrofoam container with the lid and secure with a rubber band.
Enter the strain in the "Worms" database in Quartzy.
After a week or so, a test thaw can be done. Use the small styrofoam carton in the bottom of the -80°C with a metal block on dry ice to hold your agar-frozen worm strains. Take the cryovial to be thawed and place it in the metal block for transport to the bench. Rub the vial between your palms for 2 seconds. Heat a metal spatula in a flame and rotate it around the outside of the cryovial until you can free a chunk to place on a 10 cm plate. Place the chunk on the outer edge of the plate — keep the melted worms to the outside, as you want them to crawl into the OP50. Put the cryovial back into the -80°C along with the metal block. Put the plate at the desired temperature for the strain. Check after ~48–72 hours: if you see gravid animals and embryos, the strain survived. If not, try freezing again but start with more 6 cm plates. If the strain has a transgenic marker, check that the thawed stock still expresses it.
Move four tubes to the next available position in the Worm Stock Boxes, and one tube to the next available position in the Liquid Nitrogen Stock boxes (check Quartzy for the last strain placed). Put a space between each strain you freeze.
Working stock box