A Method for the Cryo-Preservation of Acrobeloides (Cephalobidae)

Introduction:

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).

1. Transfer adults to newly seeded plates and maintain cultures at room temperature for ten days until growth is high and peak density is reached. Make sure the density of adults in the culture is high, as they survive freezing better than juveniles (unlike the C. elegans protocol that works mainly for larvae).
2. Keep cultures at 15°C for about 15 days. This slows down their metabolism and may starve them to a certain extent.
3. Wash nematodes with M9 Buffer from plates into 15ml plastic centrifuge tube and keep them at 4°C for three to four days. This starves them further.
4. Centrifuge at 2000RPM for three minutes. If the culture is dominated by adults, they tend to settle fast and a shorter centrifugation time can be used.
5. Pipette off supernatant leaving about 0.5ml (with nematodes) at the bottom.
6. Heat-shock them by leaving them at 37°C for 45 minutes.
7. Add 1ml freezing solution, mix gently but well and transfer into 1.5ml freezing vials, close and immediately keep them cool in ice. These nematodes seem not to like the freezing solution; therefore the speed of cooling and freezing are critical. The longer one leaves nematodes in the freezing solution, the fewer nematodes will recover from freezing.
8. Put them in a Styrofoam (closed on the bottom but open at the top) and freeze them (at -75°C). Fast thawing is best for survival of nematodes. Keep vials at room temperature but hold them by hand to facilitate thawing. Shake them and pour out into a petri dish half-filled with M9 Buffer before they defrost completely in the vial. Slow thawing (first keeping frozen vials at 4°C until they defrost) decreases the recovery of Acrobeloides strains we tested.

 

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.

 

Additional observations:

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.

 

Reference:

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.

 

Acknowledgement:

The study was part of a project funded by the Natural Environmental Research Council (UK).