Dear colleagues,
I am performing C elegans longevity assays using N2 as background. At about 5-6 days of adulthood I noticed a high percentage of internal hatching of larvae with consequent death of these worms. I decided to exclude these animals from the analysis since I cannot infer whether the worm's death occurred before or after larvae hatching. Moreover, I noticed sometimes body tremors in some older animals (10 days old or even more) after touching them with the platinum wire. They seemed almost dead (or already dead because pharynx nor head did not move). Have you ever experienced something like that? If so, how do you consider these worms in your analysis?
Thank you in advance for your technical support!
You see bagging (internal hatching) about 5-6 days and at a high percentage. A standard temperature is 20 oC and maturation would be 3-4 days. N2 does not have a high rate of bagging. If bagging starts at that temperature, it would start around 4-5 days. It sounds like bagging is somewhat slower and at a higher bagging, I would request a new N2 strain from CGC. When N2 is maintained too long, they tend to get additional mutations. But all those assumes 20 oC. If 16 oC, it could be about right but still bagging should be negligible. You should explain the conditions, plate or liquid, what type, temp, others would not be able to help on the community.
I am not sure about body tremors, as N2 should not show such abnormality. This is another reason your strain get mutated.
"touching them with the platinum wire" means you are using a plate assay but not liquid. The method may be tricky. Once they die, they should decay in a few days- that way you know the work was dead.
I have many researchers who were at their early stage of learning the technique, they tend to kill worms by touching. I would not trust any data in their first month. You could monitor the stability of your data. Touching should not be strong.
Thank you Shin Murakami for your kind answer.
I performed many longevity assays using the fer-15 background at 25 °C, so I didn't have to move adult worms to different plates during the fertile period.
I have already seen bagging in worms, but only in very crowded and starved plates. I didn't expect to see this phenomenon with fed worms.
Concerning the tremors, I haven't observed them in wild-type worms, but only in 2 lines of the same mutated strain which were obtained by knocking out a gene of our interest by CRISPR technology, followed by two backcrosses with N2 to eliminate possible off-targets effects.
I'm performing experiments at 20°C, with OP50 bacteria on NGM plates. Generally, when worms stop to move on plates, I gently touch them on the head to detect any spontaneous reaction.
I have planned to repeat the assay using fresh worms, in order to exclude phenotypes due to long periods of cultivation.
Do you have additional suggestions?
Thank you in advance.
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