STANDARD OPERATING PROCEDURE
Title Population Synchronization of Worms by Bleaching
Short Title, ID Bleaching Worms Created on Dec. 26, 2021
Status ** Related SOP **
Safety Instruction ** Category **
Author Nazli Hassanpour, Technical Assistant Approved by **

PURPOSE/ABSTRACT:

To synchronize populations of C. elegans by bleaching to obtain individuals in the same life stage. The same procedure likely works for C. briggsae and C. tropicalis.

BEFORE STARTING:

Ensure that your plates have many gravid (pregnant) hermaphrodites or unhatched eggs as only the eggs will resist bleaching. There is an average of 10 viable eggs per hermaphrodite.

EQUIPMENT/REAGENTS NEEDED:

  • Plates of C. elegans

  • Bleaching solution

  • M9 solution

  • Plastic transfer pipettes

  • 1000 mL pipette

  • Eppendorf centrifuge *add model

  • Rotator

  • Vortex

  • Filter tips

  • Timer

Bleach Solution 2X For 10 mL
Bleach* 4 mL
Sterile Water 3.5 mL
NaOH at 1M 2.5 mL

*Bleach: sodium hypochlorite + 5% active chlorine (keep stored in 4C fridge)

PROTOCOL

1.     Make the 2X Bleach Solution. This should be made fresh right before bleaching every time. What you don’t use can be used for spot bleaching in the future.

2.     Label tubes for each strain on the side of the tube and lids to prevent cross contamination between strains.

3.     Wash the plates with M9 solution – pipette 1-2 mL of M9 across the plate to loosen worms and eggs that are stuck on the bacterial lawn.

4.     Move the plate in circles on the bench to loosen worms more.

5.     Optional: pipette the same liquid and re-dispense on the same plate to dislodge more worms.

6.     Using the same pipette, collect the M9 solution containing worms into a 15 mL Falcon tube.

  • Pipette gently to avoid collecting bits of agar together with the worms

7.     Top-up the tube all the way with M9 solution

8.     Centrifuge at 1500 RPM for 2 minutes to collect worms into a pellet and separate bacteria/debris into the supernatant (make sure the centrifuge is programmed to stop spinning slowly to prevent dislodging the pellet *soft brake program).

9.     Discard the supernatant with a plastic transfer pipette being careful not to disturb the pellet

10.     Repeat steps 5-8 two more times.

11.     Fill the tube up to 4 mL with M9 solution.

12.     Add 4 mL 2X bleach solution to a total of 8 mL for a working concentration of 1X bleach.

13.     Vortex with full power for exactly 4 minutes – ensure that an actual vortex is observed (e.g. tornado formation in the tube)

  • If exceeded, eggs may be damaged

14.     Immediately after vortexing, top-up the tube with M9 solution to dilute the bleach as it is harmful

15.     Centrifuge at 2500 RPM for 2 minutes to pellet the eggs

  • Pellet should be yellow if the egg releasing worked (check under microscope if not as the worm may not have lysed)

  • Don’t need soft brake program on centrifuge here/brake at top speed to remove bleach as quickly as possible *add step before if it needs to be programmed each time

16.     Discard the supernatant by pouring it out as pellet is more secure now

17.     Top-up the tube all the way with M9 solution and shake to break the pellet.

18.     Repeat steps 13-16 five more times or until you can’t smell bleach

19.     After the final wash, top-up with M9 solution.

20.     Attach the 15 mL tubes to the rotator and rotate in the 20C incubator for 1-3 nights.

  • The eggs will hatch and enter diapause -> stay in L1 stage, synchronizing step

  • Rotating prevented hatched L1 from feeding on debris and exiting L1 diapause

21.     To refeed worms, remove tubes from the rotator and spin down in the centrifuge (2 min at 2500 RPM).

22.     Pipette off supernatant and top-up with M9 solution. This washing step removes secretions from hatched larvae.

23.     Spin tubes again for 2 min and 2500 RPM

24.     Pipette off M9 solution depending on how concentrated you want your worm solution to be.

25.     Break off the pellet by tapping on the tube.

26.     Transfer worms to a new seeded plate with a glass pipette and rubber bulb.

  • Don’t use plastic pipettes or tips as without the bacteria on the worms, they will stick to plastic easily.

27.     Incubate worms in the 20°C incubator to allow development.