I am growing worms in 96-well plates, in liquid medium, and it seems that the growth is not homogenous in the plates, with a better growth on the outside of the plates. I incubate the plates in the 20°C incubator, on a vibrating shaker. I use porous seals to allow gaz exchanges. I used to put a lid on the plates but I don’t do anymore. My guess is that the problem is probably that the oxygenation is not homogenous in the plate but I don’t really see how to improve it!
Does anybody have any tips or any idea what the problem could be and how I could solve it?
i was also trying to grow the worms in liquid medium (20% BHI + 80% M9) but i failed to as the worms died in 3 days
may i know what liquid medium that you’re using ?
by the way, about the aeration:
Quoted " The three critical parameters for growth in microtiter wells are 1) amount of bacteria added, 2) adequate aeration, and 3) growth temperature. If too much bacteria is added the worms will die; if too little bacteria is added the worms will produce few progeny before the culture starves. With the proper amount of food, 1000–2000 progeny will be obtained from the 20 worms added to each microtiter well, and these progeny will starve as L1, L2, and L3 larvae 4–5 days after the culture is started. Inadequate aeration causes the cultures to grow slowly or not at all. Cultures grow slowly at temperatures below 20°C or above 25°C. "
Germline proliferation and its control, by Judith Kimble§, Sarah L…
I’ve tried M9, S-basal and S-medium, but the one that works best for me is S-basal.
I’ve never worked with BHI. What does it contain?
I’ve noticed that the amount of bacteria is actually critical, but I’ve found the good conditions (after lots of tries!) so I don’t think this is the problem in my case. I growthem at 20°C in an incubator, so the temperature is stable.
So my guess is that the problem is aeration for me (and it is quite consistent with the article you quote), that might not be homogeneous in all the wells of the microtiter plate. Because in some wells, the worms grow well but not in other. The amount of bacteria and worms is quite homogoneous as I use a replicator and a multichannel pipette.
Do you grow your worms in a large volume? What stage of worms do you start with?
I experienced a similar phenomenon, which I solved by growing worms in S medium (0.5 ml) in deep-well plates, using a vibrating plate holder in a humid incubator with no lid on the plate. Air-borne contaminants can be prevented by adding antibiotic and antifungal compounds. The wells are deep enough that I haven’t had a problem with aerosolization causing cross-well contamination (by worms or bacteria). You can test your setup by pipeting worms w/ bacteria in alternating wells, and clean medium in between.
the liquid medium used in Breger J., Fuchs B.B., Aperis G., Moy T.I., Ausubel F.M. and Mylonakis E. (2007) Antifungal Chemical Compounds Identified Using a C. elegans Pathogenicity Assay. PLoS Pathogen 2007, Vol 3, Issue 2:168-178 was fairly simple, which is 20% of BHI (Brain-heart infusion agar) + 80% of M9 buffer during the screening assay.
by the way, what is the difference of using S-basal instead of S-medium ? because from what i read from Wormbook procedure, it states to use S-medium which consist of S-basal…
regarding the amount of bacteria, how do one determine how much of bacteria to be loaded ? just simply based on self-experience or there is an basic line to do so ?
i havent success in growing the worms in large scale using liquid medium yet, but i will try the Wormbook procedure as soon, hoepfully it will come out with something good. Since i am doing drug screening assay, so i always use Young adult stage sterile worms.
May i know what is the rpm during the shaking of multi-wells plate ? i remember some guys telling me it’s ok to cover the plate with parafilm with some small holes poked, however, since that i havent do on well plates yet, i cant give u certainty onto that.
thanks for your reply. Unfortunately, I can’t use deep-well plates as I read the plates on a COPAS-profiler thet is adapted for normal 96-well plates but not deep-well plates. I’m using a vibrator plate holder as well, and I don’t put a lid on the plates, only a porous seal (that is supposed to allow gaz exchanges). I don’t now if the seal could make a difference…
for the medium, some protocols use M9, some S-basal, some S-medium. The original protocol I used was in M9 medium, and I try as well S-basl and then S-medium. For me, I saw some improvements with S-basal and no more improvements with S-medium, so I now use S-basal.
For the amount of bacteria, unfortunately, I think the best way to determin it is self-experience! I’ve tried different quantity of bacteria. If you use too much, the medium stay very trouble even after few days and you could see that the worms are dead at the bottom. If you don’t use enough, the medium is cleared very quickly and the worms are starved.
guess i have to try for few times more then i will have some experience in supplying the bacteria to the medium. :-[
from my last few attempts, i observed there were some jelly-like clumps in the medium, which i suspected that it’s the bacteria clump which killed my worms in such a short while.
by the way, is there anyone here that is familiar with COPAS / using COPAS ?