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Synchronizing Worms

Synchronizing Worm Cultures


Synchronization is based on the fact newly hatched larvae will live but not develop if deprived of a food source. An asynchronous population of embryos is isolated and then allowed to hatch in the absence of food. Once all the embryos have hatched into L1 larvae, the now synchronous population is transferred to medium with food and the population of larvae resume their growth and development.

Materials:

M9:

22 mM KH2PO4 42 mM Na2HPO4 85.5 mM NaCl 1 mM MgSO4

alkaline hypochlorite solution:

approx 1% NaOCl (=140 mM) 250 mM KOH

sterile culture dish or 24-well plate

Procedure:

  1. Collect gravid adults
    • Harvest gravid adults by washing worms off well populated plates with ~1ml M9
    • Collect in 15 ml centrifuge tube or in eppendorf tubes depending on scale
    • Pellet worms by centrifugation 20-30 seconds at 150-200 x g (30 seconds at 1200 rpm in clinical centifuge)
    • Remove supernatant
    • Wash once with equal volume of M9
    • Remove supernatant
  2. Isolate embryos from adults
    • Add 1-5 mls of FRESH hypochlorite solution to worms (1 ml for eppendorf or 5 mls for 15-ml conical tubes)

      1 ml :

      700 uls H2O

      5 mls :

      3.5 mls H2O

      200 uls 5% NaOCl

      1.00 mls 5% NaOCl

      100 uls 5M KOH

      0.5 mls 5M KOH

    • Mix well by inversion and monitor progress under the dissecting scope
    • worms will disolve in the bleach solution leaving the embryos which are more resistant to bleach
    • When embryos are released (3-7 minutes) collect by centrifugation 20-30 seconds at 150-200 x g
    • Remove supernatant
    • Wash 4X with equal volume of M9
    • Resuspend worms in small volume M9
  3. Hatch off L1s
    • Transfer embryos to culture dish or well of a 24-well culture plate with sterile glass pipette
    • Allow embryos to hatch overnight (>12 hours) at 20 degrees
    • Titer L1 population by counting "swimming" L1s in 10ul spots of 10-fold dilutions
    • Transfer desired amount of worms to medium with food source
    • Allow development to desired stage

 

Notes:

 

compiled by Chad Rappleye

Aroian Lab Protocols