Does anyone have any tricks to keeping small numbers of worms (less then 500 and all of them precious) in plastic eppendorf tubes throughout multiple wash steps of a staining procedure? 500 seems like too many to pick individually for the number of washes involved, so I am looking for any tricks you may have discovered. I am currently spinning and then aspirating by pipette while viewing under a dissecting scope and although it doesn’t seem as if I am sucking the worms up, I must be, because I have huge losses. :-[
You might try only handling them with glass: glass test tubes and pasteur pipets. A small glass test tube fits snugly into a 15ml conical centrifuge tube, and can be spun safely on a low speed table-top clinical centrifuge, or of course if you’re patient you can wait for the worms to settle by gravity. Great care must be used in aspirating the supernatant because there’s not much “cling” of the pellet of worms to the glass, but other than that I can get very high recoveries of precious worms this way. I’ve used volumes on the order of 100-200 microliters in this way many times.
worms stick nicely to most tubes/tips. You can get low retention plastics or siliconized tubes/tips. alternatively you can pre-wet the tips and the tubes, then they stick much less. this works best if your worms are mixed with bacteria (for example when you wash them off plates with OP50). presumably the bugs block the walls so the worms don’t stick as much.
I’ll give those ideas a try, and see which suits the procedure (and me). It seem like such a simple thing to be having a problem with, I guess it was never an issue until dealing with limited numbers of precious worms. Thanks for your help.
Actually, we wash and bleach a lot in our lab and if you use 0.05%Tween-20 in your M9 or PBS this totally prevents sticking in our hands. I guess Triton would work as well, but you want to go for a mild detergent. We use 15ml Corning tubes to do the washes and have no loss of worms or negative effect of the Tween-20 on development.
Hi, I agree with the idea of using 0.1% Tween+ S-Basal/M9. I regularly have to wash 200-250 worms of the fresh OP50 and I think this tween+S-Basal seems to do the trick with minimal loss of guys ;D. Hope this works for you
i have another question about the procedure after the washing step.
i am using M9 buffer during the washing steps, and my problem is, how do i transferred the washed worm on to the plate ? as i was doing screening for natural products that will cure the worms from bacteria infection. i wonder how would the M9 buffer carried over to the plate give effect to my screening…
any ideas/ suggestion would be greatly appreciated in advanced