Worm Breeder's Gazette 10(1): 140
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.
1. Preparation of fer-1 dauer larvae A temperature sensitive sperm defective mutant, fer-1 (hc-1) is grown at 20 C on 150mm NGM plates with 10 ml of whole egg homogenate ( 50 ml H20/egg) dried on top. Leave the plates for 10-14 days (or even longer - but the number of dauer begin to decrease) by which time most worms on plate are dauers. Wash worms from plate with M9 or H2O and preclean either by multiple washing or by filtration through a 44 micron Nytex screen. The worms are further cleaned by flotation in 35% w/v sucrose or if only dauers are required by 1st treating with 1% SDS for 30 minutes and then sucrose flotation. 2. Adult fer-1For each 150 mm NGM plate, add 10 ml whole egg homogenate plus 1-3 g of E. coli and dry onto plate in sterile hood. Once the plate is dry or semi-dry, add 0.7 ml of packed fer-1 dauers and incubate at 25.5 C for about 2 days. This is the non-permissive temperature and necessary for a synchronous shift out of dauer hood. After 2 days, worms are ready to harvest; by this time greater than 95% should be young adults full of oocytes. Worms are cleaned by 35% sucrose flotation. The yield from 10-12 150mm plates is about 60-80 ml packed worms. 3. Preparation of oocytes The clean worms are given a final wash in egg salts buffer (esb=110 mM NaCl, 4 mM KCl, 5 mM CaCl2, 5 mM MgCl2 and 5 mM Hepes pH 7.2). Resuspend each 10 ml of packed worms in a 50 ml tube with 40 ml of esb and sonicate on ice. We have a Braun Sonic 2000 with micro and medium probes. If micro probe is used, the settings are 60-80 watts for 30- 40 sec. and if the medium probe is used, the settings are 40 watts for 30-40 sec. You can test your probe by sonicating worms at various power settings and analyzing the sonicate - too high destroys oocytes - too low just tickles worms. After sonication, the worms are allowed to settle and the supernatant removed and stored on ice. 10ml of esb are added to the worms and mixed, the worms are allowed to settle again and the supernatant saved. The settled worms are again resuspended in esb to 40ml and the sonication and settling procedure repeated 4-6 times. The supernatants from all the sonications are centrifuged at 500 xg for 5 minutes. The pellet is taken up in 10 ml of cold esb and transferred to a clear 15ml polystyrene tube; the supernatant is discarded. By this time, the oocytes are at sufficient concentration that they aggregate and settle faster than the contaminating worms. Let the suspension settle and remove the supernatant. Repeat this procedure at least 20 times (nobody said this was elegant) or until no more worms can be seen. You may have to break up the oocyte aggregate a few times to get rid of all the worms. The yield from about 60ml packed adult fer-1 is about 1ml oocytes. 4. Purity check of oocytes Presence of worms or developing embryos can be detected under polarized light for their gut granules. Usually less than a few percent of the preparation shows gut granules.