Infection model with Pseudomonas aeruginosa

Hi,

I am new around here and have just started working on C elegans last month. I have some questions on using C elegans as infection model with P. aeruginosa. As I am a complete novice in the field of C elegans, I will greatly appreciate any help! Thanks!!

My main sources of reference are here:
http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
http://www.ncbi.nlm.nih.gov/pubmed/9892699

I am intending to infect C elegans (N2 wild type strain) with PAO1 P. aeruginosa. However, I found that the infection was not successful. Furthermore, I seems to obtain very few worms at the end of my protocol.

Briefly, my protocol is as follows,

  1. Wash worm off NGM plates and treat with bleach:NaOH to obtain synchronized eggs
  2. Culture overnight in M9 buffer at 16˚C
  3. Spin down and resuspend worms in S basal and add in P. aeruginosa pellet.
  4. Incubate at 16˚C for 3 days.
  5. Spin down and move worms onto agar plate, then observe with dissecting microscope.

My questions are,

  1. Is there any effective ways to infect the worms with P. aeruginosa? And how do I verify that the infection was successful?
  2. How do I tell if a worm is dead? I can’t seems to observe any dead worms on my agar plates. Do the worm cannibalize on the dead worms?
  3. Is there any way I can increase my yield of worms from synchronization? I only obtained about 20 worms at the end of the protocol. I believe some might be lost during the spinning down and resuspending. What is the optimal centrifuge force and time to use when pelleting the worms?
  4. Also, for maintenance of the worms, can they be stored at 4˚C?

Sorry if my questions are too novice. Thank you for taking time to read this!

There is a whole literature specifically on using elegans as a model for infection by P. aeruginosa; it’s not clear from your post that you’ve looked at these papers in detail. My recollection is that within that literature PA01 is used for fast-killing, and PA14 is used to infect worms and look at the resultant slower toxicity. I haven’t looked at that literature recently enough to remember in great detail; I believe the media used is important (it determines the growth phase of PA01 and whether it does fast-killing), and the experiments I recall grew animals on plates, rather than doing a prolonged exposure in liquid as you describe.

Some other notes:
0) You appear to be proposing to do liquid culture, or at least a three-day incubation in liquid. I’d probably recommend that you start by growing worms on plates (it’s easier, for one thing, and I have no idea at all how happy your bacteria will be in liquid culture). Your liquid culture protocol appears to be S basal plus food, which does not conform to the standard protocol, and you don’t mention doing a control with E. coli as a food source.

  1. I don’t have a very good detailed recollection of the P. aeruginosa literature, but I’d suspect you could use GFP-labeled bacteria to look for infections; indeed, I think this has been done.
  2. There are comment threads in this forum about determining dead versus alive. Basically, if it is not moving, is not even pumping, and can’t be prodded into life it’s probably not alive. Other tools are available. elegans don’t eat other elegans - or at least not the cuticles - but some forms of bacteria do. E. coli is really bad at breaking down worm corpses, so they’re easy to spot and endure a long time on such lawns, but other bacteria are quite good at it. I don’t know whether corpses persist on P. aeruginosa.
  3. It shouldn’t be hard to pellet worms - 1k rcf is probably fine, and you don’t want to spin them too hard. Stage (and thus size) matters: it’s easier to pellet older worms, and easier to rinse them free of bacteria and to avoid disrupting the pellets.
  4. No, you can’t leave your worms at 4 degrees. Even 16 degrees isn’t standard (that’s 20 degrees); anything is probably OK if it works and if you’re consistent, but if you want to compare directly to the literature you should use standard conditions. Worms will grow from about 12.5 degrees (very, very slowly) to about 27.5 degrees (very unhappily). 10 degrees is supposed to cause developmental arrest and to be suitable for moderate-term storage of developmentally arrested animals, especially starved L1s or dauers; I’d recommend storing strains on clean, spotted, parafilmed plates at 12.5 to 20 degrees, and of course making frozen copies.

Hi,

Thanks for the detailed reply! That helps me a lot!

From what I read, PAO1 will kill C elegans by slow killing. However, so far, I have not been able to get the C elegans to die, nor observe any corpses (might be a problem with my observation. I am using microscope to look at the agar plate directly, not sure if that is the right way to do). I had tried using plates to infect the worms, but I found that they tend to stay at the edge of the bacterial lawn, which is why I changed to using liquid culture. But I am just testing it out, no fixed protocol that I am sticking to at the moment.

I might have got something wrong when I was using plates to infect the worms, will be glad if you can offer any tips! Thanks in advance!

Hillel makes a lot of excellent points. A few additional points/comments:

  1. I don’t know if PA01 does slow-killing. PA14 definitely does. That’s the strain that I use, and the media composition of the plates you use is important. You need to use SK (slow-killing plates). High osmolarity causes toxin production, leading to fast-killing. I’ve provided links to the two papers that provided protocols that I found clear and useful.

http://link.springer.com/content/pdf/10.1007%2F978-1-59745-570-1_25
http://www.ncbi.nlm.nih.gov/pubmed/18370168

  1. Your observation that the worms clustered around the edge of the Pseudomonas indicates that things were working. C. elegans has an aversion response to pathogens, hence the worms are either on the edge of the Pseudomonas lawn or off of the lawn. You need to assay a large number of worms, since the aversion behavior means that animals may crawl off of the plate.

  2. PA14 is very effective at dissolving worms. Corpses will remain, but they will be translucent and difficult to see. Rotating the angle of illuminate light (if your dissecting scope has this feature) makes it easier to see the corpses.

  3. In your pilot assays do you have a positive control? I would start out by testing wild-type N2 and either sek-1 or pmk-1 mutants. These are MAPKK and MAPK mutants, and the two mutants identified by Dennis Kim in his Pseudomonas sensitivity screen. sek-1 mutants should be dead within 24 hours (90% dead or more) and wild-type should be fine. These controls will let you see if your assay is working. I would also switch over to PA14, since that’s the strain I’ve seen used most frequently.

  4. Follow the time and temperature recommendations in the protocol stringently. 25ºC assay temp, grow PA14 on SK plates for 24 hours, let cool to room temp (or leave 24 hours at room temp, depending on if you use the Shapira or Ausubel protocol), use either L4s or young adults.

  5. Once you get the assay working, if you see a sensitivity phenotype, the Shapira paper has a number of good follow-up experiments to prove that your phenotype is specific and not due to another defect (ie. pharyngeal pumping, bagging, constipation).

Good luck!

Hi Jordan,

Thank you! The protocols are very useful!

I am actually trying to get PAO1 to infect the worms as positive control, which is not working too well…

Sorry that I have more questions.

  1. The dissecting microscope I am using does not have rotating function. May I know is there any way for me to accurately monitor the death of the worms? I consider counting the living worms instead, but there may be bias if the worms move to the edge due to Pseudomonas in the plate.

  2. Is there any staining agent for the worms other than using GFP-expressing feed?

  3. My model is intended to infect the Pseudomonas, follow by treatment of infection using targeted drug. However, this may take more than a week. By then, I believe the 2nd generation of worms may be generated, especially if I have to use adult worms for infection. This will probably affect the counting of worms. Is there any way I can prevent this?

Thanks!

I’m glad that the protocols were of use! I found them very helpful when I did these types of assays. I’ll just preface any comments by noting that I’ve only worked with PA14, so don’t know how assays with PA01 look. In response to your questions:

  1. Although having a dissecting microscope where the angle of illuminating light can be changed helps, it’s not essential. One way to accurately monitor the death is to increase your scoring frequency. Corpses are much easier to see right after the worm dies. With 12 or 24 hour time points, the older corpses can be almost invisible, sometimes leaving only a cuticle. Play with the light levels as well. Counting live worms is a bit of a workaround. It’s not accurate because animals can flee the plate, but it could help you get a sense if animals are dying. Once you count the live animals, that will give you an idea of how many will be dead or have left the lawn. You can then look very carefully for corpses in the lawn. With the strains that I’ve used, counting live animals gives a workable estimate of how many died, but for publication purposes you will need to count the corpses. Keeping the lawn small could also limit the area that you need to search.

  2. I haven’t seen any stains used in Pseudomonas assays to find corpses. I think that GFP-tagged Pseudomonas is usually used to see whether there are differences in colonization, not for sensitivity studies. It’s better to train yourself to identify corpses. Are you only using wild-type animals? I strongly recommend using a pmk-1 or sek-1 positive control to get the PA01 assay working and ensure that you get a good separation of Wt vs mutant. It will also help you identify corpses, since most animals will be dead in 24 hours, and many will die at 12 hours.

  3. Your proposed experimental timeline may be a bit challenging-since many animals could be dead after a week. When would you add the drug? The F1 generation would confound the experiment. Two solutions: i) pick animals to fresh plates every day; ii) use cdc-25.1 RNAi to ablate the germline and remove progeny. There is a nice protocol for doing this treatment in the Shapira paper. I think that this is the preferred solution for you, since it will also extend the survival time on Psuedomonas and give you a chance at completing a 1-week assay. This RNAi treatment works really well.

Cheers,

Jordan

Hi Jordan,

Thanks for the advice! I will be trying them out this week.

Cheers!

Hi, sorry, I need some help again…

I had modified my protocol, briefly,

  1. Synchronize the worms in bleaching solution, resuspend in M9 buffer and incubate for 12 hours
  2. Pipette part of the solution to OP50 seeded NGM plates and incubate for 48 hours to reach adult stage
  3. Transfer individual worms by worm picker onto slow-killing plate seeded with GFP-expressing Pseudomonas (grown for 24 hours)
  4. Count dead and living worms and transfer living worms to new Pseudomonas plates every 12 hours time point (this is mainly to prevent 2nd generation of worms to accumulate)

I will be getting the PA-sensitive mutants if this does not work well still.

So, may I have some comments regarding this protocol?

In addition, I am having problem with picking of worms. Previously, I used chucking method to transfer worms so I am not familiar with worm picking. I am using a 30gauge needle and mounting optical microscope for worm picking. This means that I get a quite high magnification of the worms and the distance between the objective lens and my plates is quite short. As a result I keep cutting into the agar and missing the worms. May I know are there any alternative methods to worm picking?

Thanks!!

RE worm picking, most people I know use a 1-inch to 2-inch length of platinum wire with one end mounted into some sort of handle (I melt it into the end of a Pasteur pipet, after first shortening the pipet somewhat; other people use holders for inoculation loops), with a few millimeters of the other end flattened into something like a shovel blade (using a smooth-surfaced set of needlenose pliers, a hammer and a hard surface, or applying pressure by hand with a piece of metal such as a coin that has a smooth edge). The shape of the wire from the handle to the flattened surface varies according to application and to individual taste; I usually shape the wire as a 90 degree arc from the handle to the flattened surface, with a 90-degree angle from the wire to the flattened surface so the flattened surface is roughly parallel to the handle. For other applications - such as when using an early-generation GFP dissecting scope that had a 2 centimeter working distance - the wire is essentially straight, with the flattened surface roughly parallel to the wire and to the handle. The flexibility of the platinum wire, the lack of a sharp point at the end, and the rapid cooling of platinum after you’ve flame-sterilized it should all help you to avoid harming the worm or breaking the surface of the agar. It’s usually best to scrape the bottom edge of the flattened surface along the bacterial lawn, to get a sticky mass of bacteria under the flattened surface, and then to drag this sticky mass across the worm to pick it up. You can use the pick to scoop up worms from underneath, but it can be very difficult to get the worms off of your pick. Note that when your pick is first shaped, it is likely to have some sharp edges that will soften with time, use, and repeated heating and cooling; these are a risk to damage worms and to break the surface of the agar.

Hi,

for your assay design, have a read through this earlier series of posts, some of the suggestions might help point you in the right direction.

http://forums.wormbase.org/index.php?topic=702.0

Regards

Steve