Worm Breeder's Gazette 3(2): 15
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have tried a number of different ways of growing radioactive worms including forced aeration, rotary shaking and growth on Petri plates made with radioactive sulfate. The method we like best and which seems to give a good yield of healthy worms (mutant as well as wild-type) is the following: We grow [35S]-labelled bacteria (0.5 mCi/100 ml of 0.05 mM sulfate) by rotary shaking, collect the bacteria by centrifugation, resuspend them in 15 ml of S medium and divide the concentrated bacteria between two large empty Petri plates. A chunk of worm-filled agar is added and the plates taped shut with masking tape (no apparent anoxia for up to two weeks-incomplete taping is used to prevent desiccation of stock plates at 15 C). This method is convenient for doing a large number of preparations simultaneously at controlled temperatures and the worms are in a reasonably small volume for harvesting. To maximize incorporation, it is best to gauge the amount of bacteria left by carefully tilting a plate under a dissecting microscope (precocious plates are kept at 4 C up to 24 hours). To obtain dauer larvae, the plates are harvested two weeks after innoculation. We have a modified method of harvesting worms. Flotation on 35% sucrose results in a substantial loss of worms ( especially young larvae) by osmotic shock. Flotation on Ficoli 400 of equivalent density does not work, either for reasons of viscosity or lack of osmotic shock to the bacteria. We collect our worms by spinning them at 300 x g for 5 minutes (1000 rmp in a Sorvall SS-34 head). The worms are then layered on a 14.8% w/w Ficoll 400 and sedimented for 15 minutes at 300 x g. Bacteria, worm cuticles and almost all dead and grotty alive old worms remain on top. Healthy larvae, adults and bunches of eggs are pelleted. Fungal contamination is not separable from worms by this method. If the worms are allowed to starve very much, adults may fail to sediment (not carefully examined). Dauer larvae are harvested by initial collection at 300 x g, as above, but purified by flotation for 5 minutes on 30% w/v sucrose at 1000 x g. The dauer larvae so obtained are essentially free of other worm stages and of contamination. Dauer larvae are useful to us because they are a relatively defined juvenile stage and seem to give more reproducible electrophoretic patterns of detergent-extractable proteins than mixed populations do. The patterns of detergent- extractable proteins from dauer larvae and well-fed worms show many quantitative and qualitative differences.