Worm Breeder's Gazette 5(1): 36
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.
Following the suggestion of Tony Otsuka at the worm meeting in May, we have been using chitinase to digest the shells of eggs that have been treated with NaClO. We found that the commercially available enzyme works in Egg Buffer (Hepes pH 7.2 5mM, NaCl 110mM, KCl 4mM, Mg Acetate 5mM, CaCl2 5mM); in this solution blastomeres survive and stay reasonably healthy. As suggested by Tony Otsuka, the hypochlorite treatment is required in order for chitinase to have effect; the concentration of NaClO and the length of treatment vary somewhat depending on the purpose of the experiment and on whether one starts from gravid worms or from eggs. For large scale preparations gravid worms are treated for 2 to 3 minutes with 2% NaClO with frequent mixing. The NaClO concentration is then reduced to 0.2% and disruption of the carcasses and freeing of the eggs is accomplished with the use of a homogenizer loosely fitted with a Teflon pestel. After 7 minutes in 0.2% NaClO, the eggs are collected and washed in Egg Buffer. Chitinase is added to the egg suspension. Because of the low specific activity, 5 to 10mg of protein per ml have to be used. Digestion is followed by taking samples for microscopic observation and is interrupted by washing several times with cold Egg Buffer. We have noticed that prolonged digestion damages the embryos. At this point most of the embryos have assumed a spherical shape and are still surrounded by the 'inner membrane' which is also the main permeability barrier of the embryo. To break the inner membrane and disaggregate the cells, embryos are suspended in a medium composed of one part of Ascaris coelemic fluid and four parts of Egg Buffer lacking Ca++ and Mg++. Embryos are gently homogenized (2-3 strokes) with a loose fitting Teflon pestel and Mg+ and Ca++ are restored immediately. Filtration and differential centrifugation yield fairly clean preparations of blastomeres. Cells prepared by this method appear reasonably healthy; but the fraction of them that divide a few more times has varied substantially in different experiments. Large quantities of embryos can be easily processed and the shells used as starting material for a variety of biochemical studies. In these suspensions of disaggregated blastomeres gut cells containing polarizing granules can be easily distinguished from other cells with the use of crossed polarizers. We want to instruct a cell sorter to use this property to separate E cells from non-E cells. We have used chitinase digestion on small amounts of eggs cut out of gravid hermaphrodites. In this case the 2% NaClO treatment cannot exceed 1 minute. The rest of the treatment is the same. After chitinase digestion embryos can be fixed for EM with excellent results. Eggs as early as the 2-cell stage can be fixed. We are identifying a number of ultrastructural markers of differentiation by looking at different stages of N2 embryos and plan to examine their appearance in some ts embryonic lethals. We are also using the chitinase method as another means to obtain isolated early blastomeres (e.g., Pl, P2, AB, EMST, C, etc.) to study their intrinsic cleavage pattern and their potential for differentiation and would like to confirm with this method the results obtained by the burst egg technique.