Hello worm breeders!
Just a prompt question here, worms do live if we transfer them to other agar during L4 stage or above (usually for infection model), so is it possible to grow worms directly from egg stage on other agar except NGM?
Thank you for the replies
Hello worm breeders!
I don’t have a lot of experience with this, but yes, of course there are media recipes other than NGM that will support C. elegans development. Which ones, I’m not as sure. The NGM recipe is quite simple - it’s agar, peptone, phosphate, magnesium, calcium, and cholesterol. My guess is anything that supports bacterial growth will probably support C. elegans (though maybe not happily) so long as certain needs are met. You definitely need to add cholesterol (well, without cholesterol the C. elegans should reach adulthood, I hear - but they’ll be sick and sterile, which isn’t a good result). Similarly, I know they need magnesium in defined media, though I don’t know how much magnesium is in the peptone.
Why not try it, and see? If they develop in a roughly normal time and have a roughly normal brood size, you’re probably OK. Whatever non-NGM recipe you use, you should definitely add cholesterol and probably add magnesium, calcium, and maybe phosphate.
Thank you for the replies.
We did try to grow the worms on standard Mueller Hinton agar with addition of nalidixic acid, but the embryos didn’t hatch at all and seems to be dried up.
Maybe some of the components in the recipe are toxic to the embryos since the more mature worms are able to live on the agar?
I normally avoid answering questions regarding infection like the plague, but really there isn’t that much in Mueller Hinton agar that could be a problem unless you have vegetarian worms;
so if you haven’t added the reagents listed by Hillel then there’s every chance of a non-starter when you grow from eggs. Try supplementing the agar and see if you have more success.
The older worms are probably just able to scrape through.
well, if the embryos didn’t hatch then it is probably a toxicity problem, rather than lack of some essential nutrient.
check to make sure you have the correct nalidixic acid concentration.
and what do you mean “dried up”?
true enough Eric, eggs hatch in water (most of the time anyway: http://www.wormbook.org/wli/wbg3.2p18/) and you can’t get much simpler than that, but I was thinking longer term when the eggs do hatch and the worms grow.
The ‘drying up’ observation is probably the main issue here…
If I got your problem… you cultured your worms on MH Agar with nalidixic acid and the eggs hence laid did not hatch.
well to me it appears that the worms that grew on MH agar with the antibiotic were themselves sick and the eggs were not healthy, so they appear dried out.
I would suggest you add Cholesterol to your MH Agar along with MgCl2 because it is known that
without cholesterol the lifespan and brood size of the worms is affected, and nalidixic acid too might have played some sort of spoilsport for them
like Imu rightly suggested .
Try using a lower conc. of Nalidixic acid.
one more thing… just out of curiosity… did you check worm bagging in these worms of your?
Ah, the mother of all evil is speculation…
nalidixic acid concentration: unknown
‘seems to be dried up’: meaning, unknown
location of YuuYee: unknown
Likelihood of useful answer: unk…, sorry diminishing.
Sorry for late reply, was away from the internet for a moment
Thank you for all the replies, here I shall provide some of the further details.
Together with this post, I attached a photo of comparison between the eggs which are viable and seems to be “dried up” (I’m not sure is that the proper term, the unhatched eggs look not so opaque compared to viable one)
As for the nalidixic acid, the concentration is 5 μg/ml.
About how I prepared the eggs, the eggs were prepared through bleaching protocol, and the protocol which I used is so far so good as I have no problem with unhatched eggs before this.
The eggs were NOT laid by the adults, and no problem with worm bagging as well.
Actually I’m still new with worms, still learning about them day by day.
Thank you so, so much, everyone!
well done steve. define the problem(s)!
thanks for the pictures yuu yee. the illumination/contrast is fairly different between the two images, but i think we can see well enough to get the idea.
based on the new info, my guess is that the plumber/auto mechanic would say “there’s your problem”, referring to the bleaching procedure. it can be tricky - a little too much time in bleach and all of your eggs will fail to hatch.
Yeah, the photos are not so clear. I’m sorry for that.
Hopefully still able to provide some idea
OK sure, will try out again to see if there’s something wrong with my protocol, and optimizing with all the suggestions provided here.
just following on from Eric’s observations YuuYee, what protocol are you using for the bleaching (recipe, time)? I ask because you say you have not had problems before now…
Yeah, there is no problem before this.
Lysis buffer = 2 ml of bleach, 0.5 ml of NaOH, 2.5 ml of M9 buffer.
Wash worms with 5 ml of M9 buffer, followed by addition of lysis buffer and vortex for 5 minutes.
Then spin at 1,500 rpm, 1 minute, 4 degree. Lastly wash for 2 times and spin again.
OK. There are multiple bleaching solution/protocol variations around, but essentially you haven’t got much that can go wrong…especially if you have successfully used this approach previously.
Assuming that your strains are OK, the plates are OK, the bacteria is OK etc., then there is not much left.
Are you putting eggs directly onto these plates?
Have you tried hatching them O/N and then putting the starved L1s on the plates?
If it really is down to the bleaching, then either the bleach is stronger than it was, which I doubt if it is just household bleach (being synical, I’d expect the bleach manufacturer to dilute the bleach, not make it stronger), or your conc. NaOH stock is not what you think it is…or you are vortexing too hard in the endgame.
Thank you for the feedback
Yes, I placed the eggs directly onto the plates, and let them hatch on the plates.
I just prepared the eggs via same bleaching protocol on NGM agar, and the worms are able to hatch and live well at this moment.
a couple of comments:
hypochlorite solutions do go bad over time, so different batches of bleach can produce different results.
the length of time spent in bleach can make a huge difference (there was some discussion of this previously in the forum), i.e., the difference between life and death.
it isn’t clear to me whether yuyee has done parallel plating of the same batch of bleached embryos on regular NGM and nalidixic acid medium. if this has been done and hatching was only a problem
on the NA, then that would rule out bleaching as the problem….
We are trying now.
Thank you for the replies and comments