Best ways to transfer worm onto agarose pad for injection

I am trying to do some injections. When I pick up a worm from a bacterial lawn and move to an oil drop, the worm doesn’t seem to leave the pick after touching the oil. If I try to gently brush the pick onto the pad, the worm seems to stick onto the pad in random orientations. How do you get a worm to stick its lateral side onto an agarose pad?
Sometimes, the worm does wriggle out of the pick after submerging in the oil drop. But again, the worm tends to land on the agarose pad in random orientation.

I used to have this problem a lot. What I do is pick up a few worms with a small blob of bacteria and then swirl the pick around in an area of the slide where there’s lots of the oil. They’ll come off and start thrashing. Once they are off, you should be able to pick them up individually and move them to wherever you want on the slide. I’ve attached a picture of what I normally do (this works for me, others may do stuff differently).

Here’s the slide. The red circle I drew is the agarose pad. I’ve put a drop of oil in the middle. The blue dashed line is the boundary of the oil droplet. What I do is get my blob of worms + bacteria and blot it in the center of the oil droplet until they come off. If they aren’t coming off, try rotating your pick 180 degrees and blot again. Once they’re off, take a worm one at a time and put them by the dashed line region (worms are drawn in green). You should be able to line them up in a straight line. The reason I do my injections at the boundary between the edge of the oil and the pad is that they seem to stick much better there.

I hope that helps :smiley:

I dip my pick into a little bit of halocarbon oil to coat and then use it as ‘glue’ to pick up a single worm. I then put the worm onto the dried agarose pad, and only gently try to move it around into position. I will sometimes remove some of the oil before picking up the worm so it doesn’t transfer that much liquid which can encourage the worm to thrash. I recommend picking worms that are away from the lawn, as bacteria can crud up your needle. Some people can put down multiple worms and inject them, but after a few years of this, I still find viability and injections work best one at a time.

I place several drops of halocarbon oil around the edge of the Petri plate that has my worms for injection, away from the lawn and from each other. I pick ~2 dozen worms from the lawn to off of the lawn, clean them up a bit to remove most bacteria (by flaming my pick and moving them again, say). I set up an injection pad by breathing on it to moisten it slightly and using a Pasteur pipet to pain a stripe of halocarbon oil down it. I then flame my pick, scoop up halocarbon oil from one of those drops at the periphery of the Petri plate, and scoop up most of the ~2 dozen worms that I selected and that now don’t have too much bacteria on them.

I don’t usually have much trouble at all getting the worms to come off of my pick and into the halocarbon oil on the pad. I then brush them along with my pick one at a time, getting them away from each other (not far, just not touching) and brushing down upon the worms along their axis to try to get them to stick to the pad. This is by far the most frustrating part of the exercise. I typically expect that three or four worms will not readily stick to the pad or will be damaged in my efforts to stroke them down onto the pad; these I flame. I find it’s much easier to set up a couple-dozen worms and lose a handful then to do fewer and care about each one.

When I am getting the worms to stick to the pad, I try to orient them so they are all parallel to each other (and parallel to the stripe of halocarbon oil they’ve been placed in), in half-a-dozen or so ranks of three or four worms. Their being parallel means I can try to inject their gonads in two passes without reorienting the pad for any individual worm; half the worms will have their gonads on the right where the needle enters, and when I’ve passed over the slide I can flip it 180 degrees to make another pass and get the other half. By lining the worms up fairly neatly in rows I can go from worm to worm without losing track of where I’ve been, and sometimes can save time if I don’t need to switch to the low-power objective to find the next worm.

I find it’s easier to just move all the worms you want to inject from a plate with bacteria to a clean (very dry) plate, wait about an hour (basically the time it takes to set up your injection scope, needles, dilute your DNA, etc), and then transfer them to the 1.5% agarose pads (in H20, they dry out faster in M9) in a drop of halocarbon oil. The trick to getting them off the pick is to make sure the drop you’re transferring them to is larger than the drop that’s on your pick, but no so large that they have a ton of space to float around in. Moving them to a clean plate also makes it easier to see the gonad as a lot of the bacteria will have been cleared out.
That being said, there’s a lot of different ways.