Methods

Model Organism: Worms Scientific Discussion
1

Good morning All. Please I’d like to make some inquiries about some methods am using for C. elegans presently.
1.A colleague of mine suggested to me on how to get L1 stage worms without food. So I want to confirm from the more experienced members of the worm community if am on the right track. It was suggested that once C. elegns eggs are incubated in unseeded NGM plates at room temperature. They remain at L1 stage if you don’t keep them in that state beyond 24hrs.
I would like to know if that is acceptable.
2.My second question is about counting of worms, L1 animals to be specific. I am making efforts to come up with a method of picking the very tiny worms with pipette tips, because I don’t have an automatic dispenser that can dispense an accurate amount of the worms that I want. I tried reducing the volume I set on my pipette to as low as 0.5ul before I pipette the worms from a solution they are suspended into the tissue culture plate I intend to monitor them in, but I still can’t pick close to the amount I want, say like 15 worms. I know it is more difficult with L1 worms but I need any suggestion on how I can solve this problem. Thank you for your help.

2

Good evening.

  1. Colleagues huh, you can’t do with them and you can’t do without them…however in your case you can.

    The L1 worms that hatch without food, will stay L1, arrest and after several days start to die.

    The 24 hours thing would be more important to make sure that your new worms all started (more or less) from the same developmental time point as all eggs are not equal (who said that?).

    You can leave them in liquid and the same happens, so saving on ágar.

  2. Er, you could dilute the number of worms per µL so that you can add a larger volume to the plate and hence reduce the between-plate variation. Are you adding a little Triton to ensure the critters all come out? Picking L1 worms is like trying to take your underpants off over your head…plating enough aliquots will do.

3
1.A colleague of mine suggested to me on how to get L1 stage worms without food. So I want to confirm from the more experienced members of the worm community if am on the right track. It was suggested that once C. elegns eggs are incubated in unseeded NGM plates at room temperature. They remain at L1 stage if you don't keep them in that state beyond 24hrs.
If you allow clean eggs to hatch in the absence of food - "clean" is important here, you can't pick or even rinse eggs off a plate for this - the newly hatched animals will rapidly enter developmental arrest. If you let it go long enough - typically 12-16 hours - all the eggs will have hatched and the larvae will have arrested. I don't know whether the animals "start dying after 24 hours" - I haven't looked quantitatively, nor do I recall the literature offhand. In my experience, death should be very limited for days or even weeks under good conditions - though there is a cost to remaining arrested, and as a rule of thumb for each additional day spent in L1 arrest the animal will take 1 hour longer to reach adulthood after you start feeding it.

You really should check out Theresa Stiernagle’s chapter in WormMethods for convenient, tested protocols, or you could search the forums for older posts (for example).

2.My second question is about counting of worms, L1 animals to be specific. I am making efforts to come up with a method of picking the very tiny worms with pipette tips, because I don't have an automatic dispenser that can dispense an accurate amount of the worms that I want. I tried reducing the volume I set on my pipette to as low as 0.5ul before I pipette the worms from a solution they are suspended into the tissue culture plate I intend to monitor them in, but I still can't pick close to the amount I want, say like 15 worms. I know it is more difficult with L1 worms but I need any suggestion on how I can solve this problem. Thank you for your help.
In addition to what Steve says (and regarding that, you might look at this for more information), you might find it easier to dispense eggs rather than hatched larvae. It will almost certainly be easier to count them, anyway.
4

Thank you so much for your suggestions. But concerning the transfer of the worms to wells, am using a 96 well plate and I don’t have the luxury of dispensing huge volumes of solution containing the worms. I also don’t need too many worms to monitor for my experiment.

5

And also to answer Steveh’s question, I don’t use Tritton presently when I pipette the worms.

6
  1. pipetting an accurate number of L1 is an exercise in absurdity. To reiterate what steveh said, what I found to work for me was to estimate the size of the L1 pellet in a falcon tube (say like 10ul) and working out a dilution (150ul)
    so that when I pipetted 8ul I could get roughly 100 worms. Using a wide bore tip will also help to reduce worm loss.
7

Taking advantage of the topic, I am using L1 stage without food in my screnning, and the worms spend more 48h in M9 medium without food, and in the presence of some
drugs, and they are still alive, I mean moving, after that period, but I am not sure if they are in their resistance form.

Does anyone know if is this adequate? I mean, using L1 starved for drug testing?

8
Does anyone know if is this adequate? I mean, using L1 starved for drug testing?
It seems to me it would depend greatly on the intended target of the drug candidates. There's a lot of things your developmentally arrested, foodless L1s aren't doing, so it's not a good test of drugs meant to affect any such processes.

My preference (and what I can recall seeing done before) would be that you feed your worms dead bacteria (so the bacteria can’t metabolize the drug, or be affected by the drug and become altered in a way that affects their interactions with the worms). But I suppose that for the right target, providing the drug without food might be okay.

There’s also an issue of delivery: I don’t know offhand whether starved arrested L1s pump their pharynxes and defecate in the absence of food, but if they don’t then getting the drug into their system is that much harder (and is in any case always an issue).

9

Hi,

it begs the question of what the point/aim of your experiments are? Once this is clear, as well as the knowing which ‘drugs’ you are testing then we might be better placed to give you a sensible answer.

Steve

10

you beat me to it Hillel…

11

The drugs to be tested are a library of diverse compounds, with unknown target, to have their anthelmintic action tested. I don’t have much information about it.

I have never tought about this issue of pumping the drug inside. :frowning: Does anybody know if starved L1 can pump the drug?

12

there’s indirect evidence that they might:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261173/

also direct evidence that they would:

http://www.biomedcentral.com/1471-213X/7/61

(look at the section: Pumping is an unlikely twisting force)

also background reading from Ryan Baugh’s work might also be useful:

http://www.genetics.org/content/194/3/539.full

13

Thanks a lot Steve! I’ll take a look at the papers!