Worm Breeder's Gazette 5(2): 47
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 analysis of enzyme activity we have become interested in maternally derived enzymatic activity in oocytes. Oocytes that have passed through the spermatheca unfertilized (in ts spermatogenesis mutants) can be obtained in pure form in the following manner: The double mutant CB146 and DH232 was grown on chicken egg plates by David Baillies chicken egg procedure. Synchronous cultures were obtained by isolating eggs using the sodium hydroxide sodium hypochlorite procedure of Hecht. Plates were shifted to 25 C after the L3 stage. Adult worms were collected by gently floating them off the egg plates with M9 salts and washed by collecting worms onto a 20 m nylon filter and rinsing with M9 salts. Worms were then washed in 5mM Hepes pH 7.2, 11OmM NaCl, 4mM KCl, 5mM Mg++, 5mM Ca++ and allowed to settle in a conical glass tube. After settling, worms were placed between two 6 X 10 inch Plexiglass plates and squashed at 1-2,000 psi in a Carver laboratory press. After squashing, the plates were pried apart and rinsed off with 5-10 ml buffer. The rinse was filtered through 20 m nylon filters. The released oocytes pass through the filter while the worm carcasses do not. The oocytes were then pelleted in a clinical centrifuge at setting 4 for 10 minutes and the pellets were pooled and resuspended in buffer and allowed to settle out on ice. The oocytes clump together and settle out away from contaminating debris. This last step is repeated several times by washing in buffer and is necessary to get rid of contaminating larvae and debris. These preps are currently being used to assay maternal enzyme activities (Hecht).