Worm Breeder's Gazette 14(3): 14 (June 1, 1996)
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.
Institute of Molecular Biology and Biochemistry Simon Fraser University Burnaby, BC V5A 1S6 CANADA
The normal procedure for harvesting deficiency homozygotes for use in PCR is to allow a number of hermaphrodites, heterozyogous for a deficiency, to lay eggs on a clean plate for a short period of time, remove the hermaphrodites, and wait a sufficient period of time for non-arresting eggs to hatch. Eggs which do not hatch can usually be assumed be homozygous for the deficiency. As our lab utilizes this procedure often, it became tempting to sort deficiency homozygous eggs from their heterozygous siblings by moving eggs to a clean plate directly from the deficiency stock plate (using a platinum worm pick). This abridged procedure would save time and effort by eliminating the need to set up and remove large numbers of hermaphrodites. However, subsequent PCR testing of eggs which failed to hatch after being moved in this manner soon revealed something was amiss. Although wild-type eggs (and the deficiency heterozygotes in my experiment) normally hatch with relative certainty, it became apparent that something was occurring during the harvesting procedure which was resulting in unhatched heterozygotes. Our first hypothesis to explain this was that C. elegans eggs may go through a period of development when they are sensitive to being moved. To test this hypothesis, 100 wild-type (N2) adult hermaphrodites were set onto a plate and allowed to lay eggs for 2 hours before being removed. At every subsequent hour for 13 hours, 40 eggs were removed to a new plate using a well cooled platinum worm pick. The eggs were left at 20 C for 24 hours and scored for eggs which failed to hatch. The results were as follows:
While the percentage of eggs which died at each time point generally decreased as the eggs got closer to hatching, there was still significant egg death throughout development. These results disprove the sensitivity period hypothesis (Note: We have not attempted to explain the periodicity suggested by our results, however we expect it is an artifact of our small sample size). Our second hypothesis was that eggs can be damaged by certain methods of transfer (specifically, by platinum worm pick). Again, wild-type hermaphrodites were set up and allowed to lay eggs. After four hours (the time point which resulted in the highest number of egg deaths in the previous experiment), 40 eggs were moved to a new plate using monofilament fishing line, and 40 eggs were moved by mouth pipetting the eggs directly into M9 buffer. In both cases all eggs hatched. These results suggest that eggs are sensitive to some characteristic of platinum worm picks which is not present in the other methods. The most obvious explanation is desiccation. The hydrophobic nature of the fishing line is perhaps prohibitive to the desiccation which occurs during transfer with a platinum pick. While these results are perhaps not unexpected, they provide a good reason for not departing too far from established protocols. At least, that is, when dealing with eggs.