Hi all. recently i need to capture the worms photo under UV microscope. but i am not too sure whether i am doing it correctly, especially the method to transfer the worms from 24-wells plate (liquid medium) onto the microscopic specimen slides.
what i did was:
sucks the worms from well plate using pasteur pipette onto a clean NGM agar plate
put a drop of 25mM levamisole (or tetramisole) on 1.5% agarose that already mounted on glass slide.
transfer the worms using platinum wire pick from that clean NGM onto the levamisole drop.
capture the picture.
is that the way you all do it ? i would welcome any suggestions onto this. Thanks.
Generally I add a neurotoxin, sodium azide on the agar which prevents worm movement.
Other thing is that, before mounting the worms on the slide I add a few drops of ethyl alchol on the glass slide. Then I mount around 15-20 worms on the slide (containing agar pad and ethyl alcohol).
And then carefully put a glass slide over it.
I’m usually looking at L1s in liquid in a 96-well plate. I wrote up a protocol for the Worm Breeder’s Gazette last fall, but basically, here’s what I do.
If you have enough worms in the well that the rotator makes them pile up in the middle (like when you swirl your cup with sugar or tea leaves in the bottom), you can pipet a lot of them in 1 uL of liquid medium directly onto the agarose pad. Generally they don’t stick to low-retention pipet tips unless they’ve been soaking in a lot of copper (divalent cations neutralize charges on the cuticle and promote hydrophobic interactions, I think).
Then I add a bit of levamisole, generally pipetting up 1 uL and then dialing down to 0.95 uL then touching the tip to the drop. I let the drop soak in and then add mineral oil, but that’s because I use an oil-immersion lens. You could use a drop of your medium if you have a water-immersion or air lens on your scope.
I like to draw a line of Vaseline around the pad to help stick the coverslip without smashing the worms; the surface tension really pulls that coverslip down hard.
I prefer Levamisole to sodium azide because it is less toxic. I don’t have to worry about making poisonous agarose pads, or inducing oxidative stress in the worms. (It also doesn’t scare our stockroom/purchasing guy.)
is there anything that you would put at the both slide end (lifted up a bit maybe?) so that when u lay-over the cover slip , it will not ruin the worms ?
i actually faced a problem during transferring the worms from clean NGM onto the pad. If using a wire pick, did u scrap some bacteria lawn as glue to transfer ? but would the bacteria cause the annoying background during the photo taking ? But if to pick the worms without glue, it is kind of a challenge too
Thanks for the “swirling” tips, i would give that a try
But if for my case, then i think i might need to prepare a more concentrated levamisole, so that when i add it to the worms solution (1 uL) on agarose pad, the concentration will be diluted back to 25mM.
the thing I do is that on the glass slide i make a agar pad, then on that agar pad i drop a few drops of M9 buffer. then on that i put the worms…
Buffer keeps the worms moist and thus prevent them from drying up…
and about picking worms, i also use some bacteria lawn as glue. but generally very little in amount and then pick around 5-6 worms together. it takes a lot of practice though. my advisor picks around 10worms at a time :o