Wild type worms adjust egg-laying behavior in response to food. They lay eggs when they are fed and stop laying eggs when they are starved. If starved wild type worms are put back on food, they resume egg laying almost immediately. I use the following assay to determine how well worms regulate egg laying in response to food quantitatively.
Measure the # of eggs laid by 10 worms in 30 min under three conditions:
1, starved; 2, starved then re-fed; 3, never starved.
1. Pick large dark-looking late L4 larvae, ~60 L4s/plate x 2 plates. L4s are recognized by a white crescent in the presumptive vulval region, which acquires a black central dot in late L4. Worms need to be staged as precisely as possible, because this seems to reduce fluctuation.
2. Incubate worms at 20 ºC for 30 hrs.
3. Wash worms off plates with 2.5 ml M9 buffer at a time x 2 times, combine the washes in a 15 ml falcon tube.
4. Spin down worms in a clinic centrifuge for 10 sec at maximal speed.
5. Remove supernatant by aspiration, leave ~0.5 ml liquid. (so you don't lose worms during washing)
6. Rinse worms with ~7 ml M9, spin 10 sec, remove supernatant by aspiration and leave about 0.5 ml liquid.
7. Then carefully remove as much sup as possible with a pasteur pipette, usually about 100ul liquid is left in the tube.
8. Take a 5 cm NGM plate seeded with a lawn of O.P.50 bacteria, drop on the edge of the agar 2-3 drops of 4M fructose from a pasteur pipette and rotate the plate so that the solution flows along the very edge and makes a fructose circle. Take an unseeded plate and do the same to circle the edge with fructose. You can do step 7 while you are waiting for the centrifuge to stop. The 4M frunctose creates an osmotic barrier that prevents worms from crawling off the plate and dying.
9. Under a dissecting scope, use a pasteur pipette to transfer 20-30 worms to the seeded plate from step 7. I count the # of worms transferred because I don't want to waste too many worms on this plate. Put worms down near the bacterial lawn and let the liquid be absorbed into the plate. I come back to this plate a few minutes later and move any worms that are off food back on food using a worm picking coated with bacteria. Transfer the remaining ~90 worms to the unseeded plate from step 7.
10. Incubate the worms at 20 ºC for 2 hrs. Near the end of the 2 hrs, prepare 6 fructose circled plates (4 seeded + 2 unseeded) in the same way as step 7.
11. Transfer 20 starved worms to 2 unseeded plates from step 9 with a bare platinum worm pick (no bacteria!), 10 worms/plate. (Here is my way doing it: Use a short flat-ended worm pick, press against the agar beside a worm so that some liquid gets squeezed out to lubricate. Shove the worm pick underneath the worm and pick it up. On a new plate put down the worm pick [with a worm on top of it] against the agar and squeeze out some liquid. The worm will float up and swim off. Don't tear the agar surface when you put down a worm, just make a dent.)
12. Transfer 20 starved worms to 2 seeded plates with a worm pick coated with bacteria, 10 worms/plate.
13. Transfer 20 nonstarved worms to 2 seeded plates with a worm pick coated with bacteria, 10 worms/plate.
14. Wait for 30 min at room temperate (should be close to 20 ºC). Then count the # of eggs laid on each plate. You can just remove the worms and count eggs later if you don't have enough time. Maximum of 3 strains can be assayed at a time.
15. Repeat the procedure 4 times (8 repetitions total) and average the results. For unknown reasons, there is some fluctuation in this assay that requires this number of repetitions to average out.
M9 (without glucose or MgSO4):
MRC recipe; is basically M9 for bacteria without 20% glucose. used as M9 in Horvitz lab, e.g. for egg-laying assays, everything else. Not same as M9 buffer in worm book.
Na2HPO4 5.8 g
KH2PO4 3.0 g
NaCl 0.5 g
NH4Cl 1.0 g
dH20 to 1l
M9 buffer (Worm book recipe):
also MRC recipe; Is basically M9 buffer with high NaCl to make up osmolarity for no glucose. Not used in Horvitz lab.
KH2PO4 3.0 g
Na2HPO4 6.0 g
NaCl 5.0 g
1M MgSO4 1.0 ml
dH20 to 1l