Large scale isolation of males

Hello all,

I’m interested in separating worms by sex, starting with liquid cultures of eggs taken from male-heavy plates, and I was wondering if anyone has suggestions or advice along this front. I’ve seen that Klass and Hirsh (1981 Dev Biol) and Nelson et al (1982 J Cell Bio) used nylon filters to isolate males - have any modifications to this method been put forth, or have any new methods been suggested? I’ve not found much searching through various online data bases, so I hope someone here can help. Any suggestions are greatly appreciated!

EDIT: Oh, I should add that I’m hoping to study multiple species, including male-female spp., so I’m specifically looking for approaches that don’t require specific genetic mutants.


I only work with C.elegans, but this should work for other nematodes aswell I think. I enrich my worm population by heat shock ~30°C for a few hours at L4 stage or picking males by hand and merging on a plate with only a few hermaphrodites. This will increase males in the following populations.
The nylon filtering you cite works great to separate males from hermaphrodites. This is what I do:
o Cultivate synchronized worms on plates until hermaphrodites are full of eggs and started to lay eggs! Larvae should not (!) have hatched yet!!
o Wash worms from plates and wash until cleared from bacteria/debris.
o Resuspend worms in ~0.01%Tween to M9, this will reduce loss of worms by sticking to pipette/tubes/glass.
o Pipet max 1mL (!) of worm pellet onto cell strainer (40µm) in a falcon filled completely with M9. Optionally use 30µm nylon mesh on embroidery hoop.
o Allow males to pass the mesh for ~20 minutes.
o Observe worms passing the mesh.
o Allow passed worms to settle and merge.
o Check male purity by putting a number of worms on a NGM plate.

Good luck.

Marlon’s protocol will work. If you need more detailed instructions, see Methods in Cell Biology (1995) vol. 48, pp. 278-281.


Thank you both for the replies!

I’m a new worm person and had this same question, so thank you for the replies and for posting this! :slight_smile: