Hi, I have been trying to set get a virulence assay in C. elegans going to assess virulence of several bacterial strains I am studying.
I started using N2 worms on FUDR plates as a pilot and just recently received CF512 worms which I incubate at 25 degrees after
synchronization. I use L3-L4 worms for this experiment. I’ve encountered several problems which unfortunately no one in my lab could
answer considering I was the only one using this model.
I use a binocular which was not specifically made for visualizing C. elegans and I am having a hard time finding the C. elegans on my
plates. I place 25 worms and it often happens that I can only count 20 (live or dead). Is there anything I can do to more easily visualize
the worms on my plates?
Also, is it more preferable to count dead worms,live worms or both in order to determine the survival rate?
For some reason my the plates which had OP50 as a control showed killing after 4-5 days. Most of the other plates containing
K. pneumoniae (same medium, same incubator) did not show such killing yet at such time. I use the same OP50 (spread on plates from
frozen culture, single colonies picked each time) for culturing worms and there is no problem with death of these worms on my culture
plates. What could possibly cause this killing in CF512 worms in OP50 plates?
I use 60x15 mm vented sterile petri dishes and fill them with 8-10 mL NGM and on which I spread 50 microliter of bacterial strain
(1x10^5 CFU/mL after growing to OD600=0.5) in LB. After overnight growth I place the worms and leave them on the same plate
throughout the experiment. I have noticed that the NGM does not always stay in good condition, meaning that there are artefacts or
rips in the agar after several days. Should I transfer the worms to fresh plates after a couple of days or should I be more careful
somewhere in the process of preparing medium/plates?
I was hoping someone could help me, thanks in advance!
1 and 2) re: finding/counting worms. The dissecting microscope that I use to visualize worm allows me to change the angle of the transmitted light for oblique illumination, which can help with contrast. One other point is that worms sometimes flee the plate in lifespan and pathogen sensitivity assays. You could put more worms on the plate (30-50) for your assay, and be sure to count both living and dead worms, as otherwise you are assuming any worms missing are dead, which may or may not be true. I’ve done P. aeruginosa survival assays, and it can be hard to find the dead worms, which is where the oblique illumination can help.
Short lifespan on OP50: so CF512 is dying on OP50 in 4-5 days; is this the beginning of death, or are all worms dead? If you put N2 on these OP50 plates and move them daily do the WT animals have a more normal lifespan? This could be important to see whether the issue is the plates and/or the CF512 strain. Also, since you’re using CF512 do those OP50 plates now no longer have FUDR? Maybe also check WT developmental time and brood size on your plates. If these controls are showing abnormal results really carefully check your plates.
and 2)
I have tried changing the angle of my plate (the angle of the light cannot be moved) and this indeed helps creating contract.
An increase of the amount of worms seems logical as well, I will use this in my follow-up.
The plates I use for the CF512 do not have FUDR on it and both N2 and CF512 do fine on these plates when cultured at 20 degrees.
An FUDR life span assay with N2 worms showed almost complete viability (80-90 %) after 9 days whereas at 25 degrees without FUDR with CF512
I see the first dead worms after 5 days and at 9 days only 50 % survival. At day 14 all worms have died. Looking at literature I see various experiments
varying from showing some survivors even at day 21 to survival until day 15. The trend of worms dying however appears to start later on
(around day 8-9).
Can there be that much difference between N2/CF512 or between the temperature? Would it be useful to repeat the experiments for both N2 and CF512
(with n=40 per plate)?
Hmmm, I’ve not ever used CF512 so am not really familiar with it. Maybe some other worm gurus out there have some thoughts? Are you just using CF512 to sterilize the animals to remove the confounding effect of the F1 progeny? Your lifespan seems significantly shorter than reports from the literature. You’re counting live, dead, and missing animals, right? Have you put a thermometer in your incubator to confirm temperature? If it was slightly higher, I could imagine a mutant being more sensitive than a WT animal.
Here’s what I think would be reasonable:
Check N2 lifespan to make sure plates are ok. This will let you know whether your plates are ok.
Consider another strain/condition to sterilize animals. You could use cdc-25.1 RNAi, which has worked well in my hands. (Can send you the protocol, if this is of interest)
As a bigger question, you just want to test WT worms on a range of bacteria? If WT bacteria are behaving normally in your hands, then you could pick the worms to a fresh plate 1-2 times a day until they stop laying eggs or die. This might be something that you could do now, as the CF512 data seem off.
I have checked the incubator and it shows 25-26 degrees Celsius. Could a mutant be that sensitive that fluctuations
between 25 and 26 degrees could result into this phenotype?
I have measured the lifespan of N2 worms on FUDR plates and after 9 days I still have approximately 90 - 100 % survival.
If it is possible I would much like to see the protocol with the cdc-25.1 RNAi, thanks in advance!
I use CF512 worms mainly to sterilize the worms and make sure there is no F1 progeny, there is no other specific reason.
The assay is mainly intended to assess virulence of bacteria. I could simply continue using the N2/FUDR model, considering
this appears to work (at least the control is not off).
It looks like CF512 is a temp sensitive mutant, so higher temp could cause some effect (either directly due to ts mutation, or indirectly due to the strain being slightly sick).
The cdc-25.1 RNAi protocol is from a nice Methods in Molecular Biology, vol. 415: Innate Immunity chapter written by Mike Shapira and Man-Wah Tan. Let me know if you have any questions
Place 40 gravid worms on a cdc-25.1 RNAi plate. Allow worms to lay eggs for 4 h at 25°C. Eggs laid on this plate will result mainly in Emb-laying worms.
Transfer worms to a new plate, 10 gravid worms per plate, and let lay eggs for an additional 4 h. These eggs will give rise to Glp animals.
Remove gravid animals and raise the eggs at 25°C until those develop into obviously-Glp worms (those that are not will be gravid at this point). Use these worms for subsequent assays.
If you decide to stick with the N2/FUDR model, don’t put FUDR on the assay plates with your bacteria of interest. It could inhibit the proliferation of your test bacteria.
First, I think a lot of the assays done originally with CF512 in Keynon’s group were undertaken (it is reported) at 25.5 degrees C, so probably an over-temperature issue is not where I would start looking for possible causes.
Second, you say that you have Problems with your NGM plates…they start tearing/ripping after a few days? Do you seal these plates with Parafilm or are they left open? It sounds to me like your Agar is dessicating…
More specifically, although the survival curve for CF512 compared to N2 indicates that they start shuffling off the mortal coil earlier, your survival data suggests a more fundamental problem.
At what developmental stage are you moving the worms to the restrictive temperature?
@ Jordanward - I’ll look into the protocol and try it out, thank you for the suggestion
@ Steveh - 1) It seems that the overtemperature indeed could not have been the case then
2) I saw the ripping in some plates and not all, I do seal them with parafilm.
3) I placed the worms directly at 25 degrees. Then at L4 stage I transfer them to the test plates with test bacteria. Could this alter their life-span by
growing at 25 degrees from the beginning?
The ripping you see on some plates and not on others, assuming they are from the same batch of agar suggests to me that it is mechanical damage. This is most likely to occur when the plates are seeded with OP50 and obviously also during chunking. The latter falls out as you are picking worms onto the plate.
So, perhaps you are just being a little too vigorous on some plates?
As to the second issue your CF512 (Logan’s Run) strain. In the article below, they describe a slightly different protocol for avoiding the complications of progeny on their plates, albeit that they are not studying virulence effects.
p.6103
CF512 ( fer-15(b26)II; fem-1(hc17)IV) animals are heat-sensitive and sterile and were routinely grown at 15°C. To avoid progeny, CF512 worms were hatched at 20°C and L1 larvae transferred to 25°C for 48 h and back to 20°C until harvested.