I have cultured the phenotype of N2 for some researches in NGM, but recently the worms is still spawning without egg incubation. :’(
without larvas can we observe, but there are embryos in the eggs under the high-power microscope.
this kinds of phenomena has already been twice, but inconsistently.
we test the conditions of culturing: 20℃, GOOD humidity, normal nutrition as the direction of the WORM STARTER KIT , NO infection of other bacteria or fungi expect the OP50 the null pathogeny food for the worms.
because we cant find the root of the problem, we want to listen to your advise about this problem or who have the same experience during researching on wrom.
Thank you all the same. In fact, we just want to know the elements or factors which can affect the incubation of the worms.
The worms are inability to hatch. We have tested such factors mentioned in the context, but we can’t find what kind of devil played tricks. We are exhausted! There must be something or situation ignored. So we want to know anything about why the eggs can’t hatch or what can chang worms’ lifecycle.
If the temperature, humidity and OP50 are all OK, the most likely cause is a problem with the NGM. This could be, for example, a lack of cholesterol, a very aberrant pH, etc.
Im not sure if this can help, but another factor is oxygen. Under anoxic conditions (low oxygen) the embryos arrest and they dont hatch. If you have too many worms in you culture and not much air inside that could be the problem
Thank you very much! we have test the factors mentioned, but unfortrunitely the ghost is still here. Now we focus on the PBS and NGM. Prey for us, thanks again.
When we tested the PBS, it showed PH as 5.6 almostly. however we make a mistake on PBS, I don’t think it would cause so big influence. And then we change new cholesterol agentia. The recipe is from a papper:
B. NGM Agar
NaCl 3 g
Agar Bactoagar 17 g
Peptone 2.5 g
Cholesterol (5mg/ml in EtOH) 1 ml
H2O 975 ml
Autoclave. Then, using sterile technique, add the following and mix.
CaCl2 1 M 1 ml
MgSO4 1 M 1 ml
Potassium phosphate 1 M pH 6 25 ml
Cool down slightly and pour about 5-7 ml molten agar into 6-cm petri dishes. Allow 1-2 days
to dry before use.
Intrestingly, yesterday we found there were some small worms hatching out which were lay four days ago. We wonder whether the factors influencing hatch were costed during these days, since creatures always have that kind of ability to endure bad circumstance untill the depression gone. But we still can’t find the problem.
I am glad to listen to your advice, Thanks.
PS: I am a senior undergraduate, a new soul, so I am glad to know anything about the knowledge about how to culture worms.
I don’t know if it makes any difference, but the recipes I’m familiar with all say to add the cholesterol after the media has been autoclaved, not before, waiting until the media has cooled to ~60 degrees Celsius (basically, cooled so you can rest the side of your arm on the outside of the flask).
Oh, yeah, I think it would be reasonable to consider the time & tempreture of adding cholesterol. maybe there are some change during High Temperature Disinfection. But will it be? The choleterol should be thermotolerant, isn’t it?
Thank you!
