Caloric restriction in liquid culture

       Hello everyone, I've encountered a problem with performing CR in liquid culture. I really need your advice.

Most protocols currently apply the OD600 = 1.5 for AL condition and OD600=0.15 for CR condition. In addition, they use 12-well plate to check lifespan of C.elegans with 15 worms/ml and FUDR adding.
In our lab, we need a large amount of C.elegans cultured in CR condition. Therefore, we used 500 ml flask to culture worm with the same ration 3750/250 ml. We used nylon filter to separate eggs and progeny from adult worms,
instead of FuDR ( i can’t use FuDR for my project. FuDR affects the outcome of experiment). However, under CR condition (OD600=0.15), I observed a lot of bagging worms. As bagging is a sign of starvation, I guess C.elegans went to
starvation condition; then they could’t lay eggs and died of the bagging. At this point, I wonder whether OD600=0.15 is really CR condition or Starvation condition? Please give me some ideas in this case!

I think that I would start out by testing the CFU/mL (Colony forming units) of your solution just to verify that you’re getting 10^8 CFU/mL for CR and 10^9 CFU/mL for AL. I’m not sure how much I’d trust OD readings without verifying them by plating dilutions and counting # of colonies first.

Check out this paper for more info.

Thank you very much for your suggestion! I will give it a try!