Dead or alive?

Model Organism: Worms Methods & Reagents
1

Hi
Is there an easy way to identify if a worm is dead or alive?
I am running Killing assay in which I use worms to find out the degree of virulence of bacteria. It is sometime extermeley difficult to see if a worm is dead or just not moving without a fysical stimulation.

Regards
Reza

2

I’ve heard some longevity/aging researchers monitor pharyngeal pumping to determine if an animal is alive or not: when the pharynx ceases to pump the animal is likely dead. I believe some people have used non-responsiveness to touch as well as a marker for death. I’m not a longevity researcher though so perhaps someone else should confirm this.

3

Yes! It is true and this exactyl what I am doing but the problem is that it is impossible to do it for many plates and for a long time.

Regards
Reza

4

the people who make the COPAS C. elegans biosorter seem to suggest you can use SYTOX Live-Dead dyes to stain for dead worms (REF). however, i don’t know how quickly C. elegans becomes permeable to SYTOX after pharyngeal pumping has ceased (or if it happens concomitantly with cessation of pharyngeal pumping). if you test the correlation first, maybe it’ll work for you. (maybe i’ve missed a paper that used SYTOX to assay death.)

just a thought. goodluck.

5

I’ve been doing longevity experiments for a few months, and while I’m no expert I’m starting to get fairly efficient. Frankly, it just takes practice. The experiments I set up usually have 1000 or so worms, with all the controls/genotypes/conditions/replicate plates. It’s pretty easy at the beginning of the experiment, when everyone is alive and active. It gets a lot harder around the mean life span, because so many die so fast. Still, even then it takes me less than three hours to scan them all. Pharyngeal pumping isn’t very good (at least with aging), since there’s a few days at the end of the life span where worms will still move in response to touch, but will only pump intermittently if at all.

Here’s the step-by-step of how I do it:

  1. Scan for “dead looking” worms at low magnification. They won’t be moving (obviously), but they’ll also have a very different posture. Some dead worms will go into a sort of rigor mortis, and will be stiff and straight. Other dead worms (dead longer?) will be shrivelled up. If it’s got something resembling a sinusoidal shape, it’s probably still alive, but still check it out.
  2. Zoom in, look for signs of motion. Worms that aren’t moving will often still be wiggling their nose or tail.
  3. Touch the tail of the worm gently, wait a few seconds, touch the head gently, wait a few seconds again, prod it hard, wait again. If it moves after any of these touches, immediately leave it be and look for more dead worms. I’ve noticed that really old worms will almost respond like a shrinker unc after that first tail touch.
  4. Pick off the dead worm and record it.

Examining each worm for signs of life takes me less than ten seconds. Just start small… perhaps just a hundred worms while you get used to it, then increase that as much as you feel comfortable.

I didn’t have any clue the COPAS could do that… will have to check that out.

6

Nice! I did not know about COPAS and we do not have it here in our department but I am going to check it out and the possibility of using that in our assay.

The problem here is that I want to see the degree of virulence of different strains of the same bacteria using C. elegans as model. Depending on each particular strain worms will live either as long as E. coli or shorter, some will die shortly (1-2 days after) after being transferred to the plate. I therefore do not want til kill or weaken the worms due to the use of any other agent/process such as staining.

Another problem is that the bacteria I use to assay (mainly Pseudomonas aeruginosa) makes a dense bacterial lawn which makes it much more difficult to find and observe the worms. At the same time worms become sick and much less motile when they are feeded on Pseudomonas.

I want to do the assay for as many as 70-100 isolates of the same bacteria so the physical stimulation of the worms will become extremely difficult. After trying out the method I started to do this for just seven strains and OP50 but I found it soon that It will take extremely long time to run the assay for all the isolates …

Thanks a lot for the comments and hope for more…

Regards

7

100 isolates is indeed a lot… you could probably do it, but it’d be many many months of nothing but lifespan assays.

There’s one more clever trick I’ve heard of, which may be of some use for you. I saw a talk by Andrew Samuelson where he described his method. Basically, he uses 24-well tissue culture plates and makes several replicas. At each time point, he’ll fill all of the wells on one plate with liquid, which will make even inactive worms start thrashing, and then continue to poke the worms that don’t move. Each replica plate is discarded after this. Supposedly this is a bit easier (at least easy enough to genome-wide RNAi screen) and gives more statistical power. It’s described in a bit more detail here, under the “life span assay” section.

Might not work if your bacteria is too thick and sticky for the worms to start swimming once flooded. Still, it’d be easy enough to try.

8

MTT-formazan has been used as a colorimetric indicator of nematode viability. Metabolic reduction produces a blue color in living, but not dead, animals. The reference is James CE & Davey MW, 2007, Parasitology Research.

Good luck,
Harold

9

If you need a reference for SYTOX Green, it’s Gill et al. Free Radical Biology & Medicine, Vol. 35, No. 6, pp. 558–565, 2003. Although they used the COPAS worm sorter to handle the worms, you could probably figure out another method. However, the worms would be in liquid, not living on agar plates.

10

Any direct method that can avoid the manual counting of the dead and live worms and make the process comparatively less laborious?

11

I am performing the Assay using the M9 Buffer instead of the NGM Agar plates. Will it be a right idea to perform an assay in the M9 buffer? As few people advised that M9 is not a good idea to perform an anthelmintic assay. Please give me your valuable suggestions.

12

It does not seem that there is a direct method.

13

Are you performing killing or anthelminticin assay in M9? I am performing killing assay and using a liquid medium will soon lead to bacterial over growth and worms will die due to secondary effects of such a growth therefor it will become difficult to find out the degree of virulence or if the bacteria is pathogen at all. By the way sorry for late respons, was on vacation.