Pick worms to six 6 cm plates for a particular strain. 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 have no bacteria left on them. The strain is ready to freeze when there is no bacteria and few or no embryos remaining. Most of the animals should be L1, L2, or dauer.
Prepare cryovial tubes with cryo-safe labels carrying the strain name and date frozen. Use the "Cryovials Label Template" for printing labels with the correct information.
- We currently freeze six cryovial tubes per strain: four for working stocks, one for long-term liquid nitrogen storage, and a sixth to use as a test thaw.
- If you want to freeze more than six tubes, prepare more than six 6 cm plates per strain.
- When the last tube is thawed, you must refreeze the strain.
When the strain is ready, use a sterile 10 mL serological pipette to aliquot 3 mL of freezing solution into a 15 mL conical tube. Aliquot ~3.5 mL of M9 into a different 15 mL conical tube.
Loosen the lids on all the cryovials.
Wash worms off the plates by pouring ~3–4 mL of M9 onto the first 6 cm plate. Shake or swirl once, then transfer the worms and M9 to the next plate, until all six plates are washed. Do this very quickly, as the M9 will soak into the plate. Transfer the worms to the 15 mL conical with the freezing buffer. The final volume in the conical should be 6 mL — add more M9 if needed.
Use a new sterile pipette to pipette the M9/worm/freezing solution mixture up and down once to mix. Be gentle and slow, so as not to introduce bubbles.
Use the same pipette to aliquot 1 mL of the mixture into each tube, replace the caps, and place the 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 at least 24 hours, move four tubes to the next available position in the Working Stock Boxes, move one tube to the next available position in the Liquid Nitrogen Stock boxes (check Quartzy for the last strain placed), and thaw one tube as a test thaw. Put a space between each strain you freeze.
Working stock box
- For the test thaw, empty the contents of the tube onto a 10 cm plate just after the contents melt. Check the plate after ~48–72 hours. If you see gravid animals and embryos, the strain survived the thaw. If not, try freezing the strain again. If the strain has a transgenic marker, check that the thawed stock still expresses it.
- As you move the strain into the Working Stock and Liquid Nitrogen Stock Boxes, write down the locations in the Freezer Log — while you are physically moving the strains.
If the strain information is not already in Quartzy, enter it: plasmids and genotypes, whether the strain was lab-made or received from another lab or the CGC, and growing temperature. After the tubes have reached their final Working and Liquid Nitrogen Stock positions (step 11), record that information for the strain in Quartzy, along with how many are in stock.
