You will need: Thick 3% agarose pad slides, pipettor and tips (preferably low-retention) for pipetting <1 µL, 25 mM levamisole*, 22 mm square cover slips, a syringe of Vaseline**, a dropper bottle of plain mineral oil, a compound microscope with an oil-immersion lens, and immersion oil.
1. Place slide on stage of dissecting microscope.
2. Pipet 1 µL of worms in liquid medium*** onto the pad, pulling the drop into a streak. (It actually works better if you scratch the pad surface slightly so the worms will stick to the scratch instead of being pulled into a single clump by surface tension.) If there are enough worms for your experiment, go to next step; otherwise, repeat until you have enough worms.
3. Draw up 1µL of 25 mM levamisole, then dial pipettor down to 0.95 µL. Touch pipet tip to drop of liquid. The worms should start coiling in seconds. Dial down pipettor 0.05 µL and repeat for other drop(s).
4. Draw a line of Vaseline in a square around the pad, smaller than the 22-mm cover slip. The line should be ~0.5 mm thick.
5. If the pad is good and dry, the drop should absorb in 10 minutes or so. The worms will usually uncoil at least partially by the time they stick to the pad. (They often glide over the smooth pad under the influence of surface tension as the drop dries.) Make sure the liquid absorbs/dries well enough that the pad is flat again; if you leave a bump, there is enough dampness around the worms that refraction interferes with focusing. Do not overdry, as this can damage worms. If it takes longer than 15 minutes for the drop to disappear and the worms look shriveled, this means the pad was not dry enough and the liquid evaporated instead of soaking in. This increases the salt content and osmotically dehydrates the worms.
6. Place a drop of mineral oil on the pad and cover with a coverslip. The oil should spread to cover the whole pad and be retained by the Vaseline seal.
7. Observe the worms on the microscope using an oil-immersion lens.
*I prefer levamisole as a paralytic instead of sodium azide because it is relatively non-toxic to users and does not induce oxidative stress in worms the way azide can, which may confound experimental results.
**The best way to get Vaseline into a syringe is to melt it in a small disposable beaker at about 45°C and draw it up into the syringe without a needle. Wipe the luer-lock end with a Kimwipe and attach the needle. Cutting off the slanted end of the needle with wire cutters makes it easier to form a line on the slide.
***I expose worms to substances in 96-well plates on rotary platform shaker, so it is easy to pipet worms from the clump at the center of the well. Worms can be picked into a drop of liquid. Spread the drop carefully with the pick for rapid absorption.
Articles submitted to the Worm Breeder's Gazette should not be cited in bibliographies. Material contained here should be treated as personal communication and cited as such only with the consent of the author.
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