Worm Breeder's Gazette 17(3): 34 (November 1, 2003)
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||Institute of Cell, Animal and Population Biology, Ashworth Laboratories, King's Buildings, West Mains Rd., The University of Edinburgh, Edinburgh EH9 3JT, Scotland, UK|
Preservation of nematode strains at low temperature is widely used. There is a general understanding that the methods hitherto developed are more applicable to Rhabditida, but less to Cephalobida. Consequently cephalobids have to be maintained in culture for future use. Maintaining cultures is time consuming and labour intensive.
We have over 100 culture isolates of Acrobeloides which we have been maintaining for the last three years. The objective of this experiment on methodology was, therefore, to be able cryo-preserve these culture isolates. All strains were maintained on E. coli (strain OP50) seeded MYOB plates. The standard freezing protocol for C. elegans was first tested (Sulston and Hodgkin, 1988), but the method did not work for our isolates. On the other hand culture isolates of a different genus, Panagrolaimus, were frozen successfully following the C. elegans protocol. Recovery was high (80-90%) for Panagrolaimus. We subsequently developed a method of freezing Acrobeloides sp which may be of utility for other nematode zoo-keepers.
Description of the method for the freezing of Acrobeloides:
Solutions needed for cryo-preservation are M9 buffer and Freezing solution, as described for C. elegans in Sulston and Hodgkin (1988).
Normally some nematodes start to show signs of movement about three hours from thawing, but noticeable body movement can be seen much later, usually 24 hours after thawing. Nematodes can be transferred to seeded plates after washing them in buffer. Recovered nematodes transferred to seeded plates reproduced well in culture.
1. Fast growing cultures of Acrobeloides do not survive freezing as well as aged ones.
2. Survival of larvae is much lower than adults. In most cases larvae die while adults survive.
3. The "Slow-Freezing" protocol described for C. elegans (Carmel L., web-published) does not work for Acrobeloides. Slow freezing according to the C. elegans-protocol requires keeping nematodes in Freezing Solution and Buffer at low temperature before freezing, but this results in the death of all stages of Acrobeloides.
4. Exposure of nematodes to heat (37°C) for longer than 45 minutes decreased recovery. But we did not investigate the effect of different levels of temperature on recovery.
5. Total density is not an indicator of degree of recovery, the number of starved adults is.
6. In all cases nematodes were frozen for 24hrs only. The effect of long term freezing was not investigated.
Carmel, L. The Effect of Freezing and Thawing Rates on Cryonic Treatment of Caenorhabditis elegans. Website address: http://share3.esd105.wednet.edu/mcmillend/00/carmell/carmell.htm
Sulston, J. and J. Hodgkin. 1988. Methods, pages 587-606. In: W. B. Wood (Ed.) The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press. 667pp.
The study was part of a project funded by the Natural Environmental Research Council (UK).