Worm Breeder's Gazette 5(2): 9
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.
In our laboratory, we routinely use dauer larvae to obtain large, synchronous cultures of L4's and adults for biochemical purposes. We have found that large numbers of dauer larvae can be obtained using eggwhite plates (a modification of the method of D. Baillie and R. Rosenbluth, WBG 2 (1)). Egg-white plates are prepared by stirring the white of one chicken egg with 50 ml of boiling distilled water for several seconds, homogenizing the mixture for one minute in a Waring blender, and layering 3-4 ml of the resultant liquid slurry onto a standard 100mm NGM plate containing a lawn of E . coli . After drying overnight, egg-white plates are seeded with about 1500 dauer larvae and incubated at 20 C . These dauer larvae develop into adults, but for some unknown reason a large proportion of their progeny develop into dauer larvae and become arrested at this stage . Approximately 1x10+E5 dauer larvae are usually recovered per plate after 5-7 days incubation time . Yields as high as 2x10+E5 per plate have been obtained in some instances. For purification of dauer larvae, animals washed from individual egg- white plates are incubated with 5-10 ml of 1% SDS for 30-60 minutes, collected by low speed centrifugation, resuspended in 0.5 -1 ml of buffer, and centrifuged through a 2 ml cusion of ice-cold 15% ficoll for 10 minutes at 300 xg. Intact dauer larvae pellet through the ficoll, while egg matter and worm carcasses remain at the interface . Because a small number of non-dauer animals sometimes escape this first SDS treatment, the entire procedure is usually repeated . Purified dauer larvae can be stored in Mg buffer at 16 C until use and retain good viability for 30-60 days . It has been our experience that dauer larvae obtained from egg-white plates recover much more synchronously than do dauer larvae isolated from starved E . coli plates, especially if they are used within 2 weeks of isolation. Also we have noted that the synchrony of recovering populations tends to decrease as dauer larvae are stored for longer periods of time.