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M/1998/3.00 Appendix 5.07


Cryopreserved organisms should preferably be stored below -135 C in order to be nearly at or below the limit for ice crystal growth (-139C). Storage temperatures above -70C are not acceptable.

Prior to storage the cells must be cooled at a rate at which production of intracellular ice-crystals is avoided. During freezing the bulk of the free water crystallizes, leaving a saturated solution of high osmotic value surrounding the still unfrozen cells, which dehydrate and shrink to obtain an osmotic equilibrium. The exposure of the cells to this solution should result in dehydration to such an extend that the freezing temperature is depressed sufficiently. The optimal cooling rate depends on the size of the cell, the thickness of the cell-wall and the cryoprotectant. Since small cells (e.g. bacteria and thin-walled fungal cells < 5 mm) can dehydrate very quickly, they can be cooled almost instantaneously but thick-walled propagules and cells < 5 mm must be cooled slowly.

For thick-walled propagules and propagules > 5 mm the optimal cooling rate has to be established by cryomicrocopy. When this can not be checked a cooling rate of -1C/min. to -40C must be applied (but for some cells slow cooling at -1C/min is harmful (e.g. oomycetes)).

Material is revived by warming it as fast as possible. Generally the cryovials are thawed in a waterbath (revival 5 min. at 30C). However, some species or groups may not survive 30C. For example, Oomycota are thawed at 25 C.

Demands on equipment

1. The vials have to be closed to prevent infection (liquid nitrogen or nitrogen vapour is not sterile).

2.. When stored in liquid nitrogen, the material should be unbreakable to prevent splintering in case of leakage and penetration of liquid nitrogen in the cryovial.

3. When taking vials out of the cryostat, precautions have to be taken that the material warms as little as possible. This may either imply not to remove the container completely or to place it in a cooled (about -70) chamber.

4. When the material is stored in the vapour phase, an automatic level controller is required as the vivostat contains relatively little liquid nitrogen. An alarm system to detect the lowest permittable level is always necessary.

5. Freezers should be provided with an alarm system and a back-up system. This may either be a connection with a liquid nitrogen container or a generator.

6. When liquid nitrogen is used, the storage room should be provided with an oxygen detector.

Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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Copyright CABRI, 1998

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