In order to get synchronous culture of C. elegans I bleach the worms and transfer the axenized eggs to 250 ml M9 buffer in a 1 litre flask . However, after incubating o/n I am not able to retrieve the L1-worms from such a big volume ( I am sure that the eggs are not damaged beacause if I seed them on NGM they hatch after some hours).
I wonder if I can transfer the eggs directly to NGM plates ( E coli OP50-free) or this could bias my experiment. Do you have a method to harvest the L1-worms from M9 buffer?
Any suggestions would be really appreciated! Thanks in advance!
Yes you can hatch eggs o/n on NGM plates and they will also go into L1 diapause. I do not imagine that it would ruin your experiment, but without knowing exactly what you are looking for or doing I cannot say that with certainty. If you are comparing two samples (e.g. N2 and mutant) then as long as you treat both the same it should be the same. An article in the most recent Worm Breeder’s Gazette (WBG) from the Reinke and Lieb groups indicates that there is few gene expression differences between worms grown in liquid vs plates (http://www.wormbook.org/wbg/volume-18-number-2/few-gene-expression-differences-between-c-elegans-grown-in-liquid-versus-on-plates/
When I did it in large volume of liquid, it is very hard to spin down the L1s. The trick to help out is to put the flask in the cold room for about 30m to get them good and cold, and then transfer them to cold centrifuge tubes and spin in the cold. So - everything cold!
I have been using the following method to great success:
After bleaching animals, I rinse with M9 3x.
Instead of sucrose floating to get rid of carcasses, I use 40 uM mesh cell culture filters and filter into 100 mL of S-medium without food (check wormbook for reciepe).
I shake this overnight (this shaking seems to be essential)
I then pour dipaused L1s into s-media containing food and continue shaking.
I do all of my shaking in non-baffled flasks, but have found that the key is to slowly turn the speed up on the shaker so you get maximum vortex made so max aeration.
Hope this helps! If you have any other questions, contact me at: tmedwar@emory.edu and I will be happy to send you all of the details!