C. elegans Bleach Synchronization

Materials

M9
15mL conical tubes
Bleaching Solution (see C. elegans recipes)
K medium or S medium depending on application (see C. elegans recipes)
Glass slide if titering

Protocol

Grow up desired number of 10 cm plates for bleaching. You want there to be maximal number of gravid adults. Each 10 cm plate yields approximately 5000 embryos. Strains vary significantly in this amount. It is easier to generate more 10 cm plates than you think you might need for synchronization.

Option 1: Chunk to 10 cm plates and monitor (~2-3 days) until you see mostly gravid adults.

Option 2: Pick to 10 cm plates 30-50 L4s and monitor (~4-5 days) until you see mostly gravid adults.
Wash worms off plates using a sterile glass pipette into a 15 ml conical tube. You can squirt M9 onto one plate of Strain A, transfer the liquid to another plate of strain A, etc. Wash all plates from a single strain into one 15 ml conical tube. It is easy to pour from the final 10 cm plate into the 15 mL conical with a little bit of practice.
Spin down worms at 1100 rpm for 1 minute using the table-top clinical centrifuge.
Aspirate off the M9, being careful to not suck up any of your worms.
Add 6 ml of bleaching solution to each 15 mL conical. Shake the tubes manually, use a nutator, or use the plate vortex if you have many strains.

a. After four minutes of shaking, check to see if the adult worms are dissolved every minute until complete.

b. Be very careful not to over bleach the worms. Once you see that the carcasses are nearly dissolved, move to the next step. If there are only embryos left, then you have gone too long.

c. The freshness of the bleach solution matters. Ineffective bleaching solution (kept at 4ºC longer than one month or kept at room temp for longer than eight hours) will require more time to bleach worms and will not dissolve the worms uniformly.
Once there are just a few adults remaining, move quickly to spin down the embryos at 1100 rpm for 30 seconds. It is best to have the reagents next to the sink and prepared for the next few steps.
Immediately decant off the bleach into the sink.
Add 10 ml of M9 using a sterile glass pipette to each 15 mL conical tube.
Invert three times as you walk back to the clinical centrifuge.
Spin down the embryos at 1100 rpm for 30 seconds.
Aspirate off the M9.
Repeat steps 8-11 two more times.
After the third M9 wash, resuspend the embryos in 2 mL of S medium, K medium, or M9 (depending on your application).
If desired, determine the titer of the embryos.

a. First count the number of embryos in 10 aliqutos of 5µL.

b. Adjust the resuspension liquid to obtain the desired concentration (e.g. 0.5 - 1 embryo per µL).

c. Mix by inverting the tube and confirm the titer by counting 10 aliquots of 5µL. Note: If the volume is >7.5mL, transfer to a 50mL conical (and ensure the volume doesn't exceed 35mL) or split amonst multiple tubes.
Transfer the embryo solution to a labeled glass culture tube.
Place all tubes to either the nutator (set to speed 13) or a shaker at 180rpm at room temperature for 12-20 hours to allow embryos to hatch.
Re-titer the L1s to determine the concentration that survived the bleach protocol and adjust according to your application.

a. To restart development, plate out onto fresh plates or feed in the culture tubes. You can pipet embryos or L1 larvae with pipet tips (~ 2k embryos or L1 per 10 cm plate).

b. For plate assays, titer the L1 solution to 1 L1/µL in K media. Note: If you see a lot of carcasses, let tubes mix and settle before drawing off L1s from the top.