Dear, my name is Rodrigo and I am a PhD student.
I work with C. elegans. I am having some difficulties of getting organisms at same size. I need of a culture with 90 to 95 % of stage L3 larvae.
I would like to know if there is some protocol for to get it, some type of synchronization or straining …I tried synchronization, but the number of obtained worms was small for my objectives. I need about de 200,000 larvae for each experiment that I will do.
I wait to help out the group
- Use 10cm or 15cm enriched peptone plates seeded with strain NA22 to increase the number of animals.
- Harvest the worms soon after the bacteria is depleted (adults retain embryos when food is limiting).
- Treat with bleach to recover embryos.
- Hatch on plates without food for 24 hours to obtain synchronous L1s.
- Transfer to plates with food.
Hi,
as well as thinking about the number of plates you will need to generate 200,000 worms for each experiment, you also need to think about the timing (logistics) of doing these experiments.
For example, do you need early or late L3s, or do they just have to be somewhere in the L3 phase? For example newly hatched worms hit L3 @ approx. +38hr and pass into L4 about 17 hours later when grown at 16 degrees C. At 20 degrees C these figures are +24hr and +34hr respectively.
Also, don’t forget to add to this calculation the fact that every hour the worms sit as L1s on plates/in liquid media without food will delay their development a little.
Last, have you thought about how you might identify that they are L3 worms? Size is one possible way, they will be ~500µm long…but it’s really worth it to spend a fair amount of time looking at L3s under the microscope so that you can easily recognise them later during your experimental work (that is how they differ from L2/L4 worms as sometimes size is difficult to judge just by looking through your dissecting microscope).
Steve
I agree, it’s very important to familiarize yourself with the different larval stages. I wouldn’t go for size as a measure of stage though, especially if you’re working with a mutant that you’re not fully comfortable with.
yes of course you’re right Snug about the size being less useful for mutants that might not grow and develop like wt, but as a general introduction to learning the stages of development (in wt) it is a useful comparison. Morphological features are more specific, but then they too could be different in your mutants to wt.
Steve
I just want to mention what I did before.
I also used bleaching method to synchronize the stage of the worms, however what I want is L4, which is easy to recognize. The problem is even ~3 days after bleaching, the stage of worms would be quite different, with some in late L4 while some in early L4 or even younger. I guess it is because of the different stage of embryoes. Anyway, what I want to say is, if you want L3, which is harder to distinguish, even bleaching could not give you exact L3 stage that you want. For the observation, I usually compare the size between L4 and other worms that would be smaller that L4 but bigger than L2. Hope this will be helpful.
Emily
Hey, I’ve never done this and don’t know if this will work, but if even bleaching does not give worms at near-perfect synchrony because the embryos themselves could have been at different stages as you suggested, then why don’t you let this generation of worms grow, pick a bunch of L4s that look synchronized, let them lay eggs for a short time, then let this generation grow, and do the L4-picking and egg laying for this and one more generation. After three/four generations you may get a synchronized population. Some worms would still be out of synchrony, growing slower or faster, but they will be only a very small proportion of the population…
Hi,
you should read the earlier discussion;
http://forums.wormbase.org/index.php?topic=344.0
Hillel Schwartz and Andy Peters covered this issue in great detail and with great clarity…
Steve