Low hatching rate of synchronized eggs by bleaching method

Model Organism: Worms Scientific Discussion
1

Hello! I have been using the bleaching method to obtain synchronized L1 for chemical exposure assay (so I use K or complete K medium instead of M9 or S buffer). However, there is a very big variation in the hatching rate of the synchronized eggs (10%-90%) while L1 yield ranges from 3000-25000. When the hatching rate is low, worms are somewhat unhealthy and dyssynchronous after further chronic chemical exposure.

I have been trying to have a consistent protocol from worm picking, bleaching to incubation. Usually, the yield of eggs is consistently between 40-70 eggs/5 uL after bleaching, but the hatching rate was just varied batch by batch (sometimes really low).

I read previous related topics and learned that I should have performed a gentle vortex instead of a continuous and vigorous vortex. I will try with gentle shaking next time, but would like to know if anyone has any other suggestion to have a consistently higher hatching rate? For all the process mixing the worm/egg pellet and bleaching/washing buffer, do you just invert or shake the tube?

http://wbg.wormbook.org/2016/05/16/c-elegans-synchronization-small-and-large-scale-protocols-to-isolate-synchronized-l1-larvae-and-beyond/
Also, I read this large-scale synchronization protocol in which they start with eight 100mm plates
(each has 15 gravid adults; 3-7 days @20oC), their average L1 yield was 2533. Based on the number of worms they’re using and my experience, the yield seemed pretty low (I set up 20 young adults on one 100 mm plate for 4 days, can obtain 6000-20000 L1). Does anyone have an idea about the yield given that scale?

Many thanks in advance!!

Below is my detailed bleaching method:

1. Worms for egg preparation:
(100 mm NGM plates were seeded with 500 uL OP50, incubate @20oC and room temperature for another 2 overnights)
	a. One 10 cm NGM plate containing 15-20 N2 L4-young adult for 4 days 
	b. Two 10 cm NGM plate containing  25-30 him-8 adult for 4 days 
Usually a lot of gravid adults and laid eggs after 4 days @ 20oC and resulting in 50-100 uL of worm pellet. N2 is usually with minimum OP50 left while him-8 mutants grow/develop a little bit slow and thus still with sufficient food.
  1. Collect worms using 10 mL of K medium in 15mL tube using glass Pasteur pipet
    3. Centrifuge @ 1300 xg, 2’00, remove supernatant
    4. Wash with 10 mL of K medium (1300 xg, 2’00) by shaking the tube for several times, remove supernatant
    5. Add 5 mL of lysis buffer (3.5 mL H2O, 1 mL Clorox bleach, 0.5 mL 5N NaOH)
    6. Perform 6 minutes of a continuous and vigorous vortex (no worms visible by eye)
    7. Add 5 mL of K medium immediately to stop bleaching reaction, mix well by shaking the tubes several times
    8. Centrifuge @ 1300 xg, 2’00, remove supernatant
    9. Wash with 10 mL of K medium, mix well by shaking the tubes several times
    10. Centrifuge @ 1300 xg, 2’00, aspirate to 100 uL.
    11. Add 3 mL of complete K medium and perform a gentle vortex shortly. Check egg number in a 5 uL drop.
    12. Incubate on an orbital shaker at room temperature (~22oC) for one overnight.
    13. Check L1 number in a 5 uL drop the next day.
2

For one, we never bleach for a certain time. We closely monitor a drop from the worms in the bleach solution and stop the bleaching process as soon as we see the worm body parts are all gone under a microscope. When you have fewer worms, the time needed is less and if you perform a certain length of bleaching it will cause more damage to the eggs (because the remaining bleach in your mixture is stronger, compared to when there are more worms for the bleach to work on).
Second is that after the bleach step, we wash the eggs for at least 3 times with clean buffer, to fully remove leftover bleach.

The bleach recipe might vary a little, but I don’t think that’s a problem. I know some labs use 1 step bleach method, and some labs do 2 step bleach, and with slightly different bleach recipe too, but they all work well.

I hope this helps.

3

I don’t know if it actually makes any difference, but I was told never to vortex the worm eggs vigorously, rather to swirl the tubes or to vortex them gently. Worth checking if you might be mechanically damaging the worms.

Also, I use a somewhat different formula (with NaOH and NaOCl rather than Clorox) and judge by eye when bleaching is done, usually circa 10 minutes.

4

I personally resuspend the worms in 3 mL S basal and add 2 mL of a 2:1 bleach:NaOH solution, then vigorously shake by hand for 4-5 minutes. I wash the worms three times after that to remove all traces of the bleach mix.

However, I think in our lab everybody uses a slightly different protocol, with different bleaching solutions and shaking times, and it seems to generally work quite well with everyone. I sometimes have difficulty with specific mutant strains, that sometimes hatch fine and sometimes don’t hatch at all, but I guess your problem is also with wild-type worms?