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LABORATORY PROCEDURES FOR MICROORGANISMS

Appendix

M/1998/3.00 Appendix 5.07.2


Protocol: Cryopreservation of fungi

Fungi are stored in sealed polypropylene straws. To handle the straws under sterile conditions use straw holders and adjusting frames as described by Stalpers et al. (1987) is recommended.

All manipulations are carried out in a recirculating laminar airflow cabinet (Biohazard CLF 406, Clean Air, Woerden, The Netherlands) to avoid contamination of the organisms and contact with the technicians handling them.

1 Preparation of straws

1.1 Polypropylene medical straws (diam. 4mm) (Polarcup Ltd. Gosport, United Kingdom; straw code ST30-184PU-JA) are cut into pieces of 47 mm, heat-sealed at one side with an Automaster sealer (AM 400, Audion Electron, Amsterdam, The Netherlands) and autoclaved (20 min. at 121C).

2 Filling of straws

2.1 The fungi are grown on the media as indicated in the CBS database on a 9 cm petridish. After incubation the identity is checked by the specialist and notes are reported in a logbook.

2.2 When the identity is correct and the culture is actively growing, 5-8 plugs of 2.8 mm diam. are punched out from the agar culture with a cork borer with pin (Stalpers et al., 1987) and transferred into the straw. 0.5 ml sterilized 10% glycerol is added to the straws with a sterile syringe and the straws are heat-sealed to close them.

Labels with the CBS accession number are glued on the upper seal of the straws with an ultra low temperature resistant rubber glue (Nederlandsche Speciaal Drukkerijen, Delft, The Netherlands). Straws are checked for leakage and if necessary resealed.

2.3 The straws are cooled at a rate of -1C/min. to -45C followed by cooling at a rate of -50C/min. to -75C in Sylab Icecube 1610 programmable freezer (Sylab, Purkersdorf, Austria).

2.4 They are stored in the gas phase above liquid nitrogen in a vivostat (Union Carbide LR320) equipped with a Union Carbide Level Controller (Type M280) or in a 135C or -140C freezer (Queue Cryostar) provided with a LN2 back-up system. Straws are stored in aluminium racks, containing 10 drawers divided into 64 squares. For each strain 12 straws are prepared; 8 are stored in an aluminium rack, of the first batch one is stored separately in the back up collection, one is opened for a viability check 24 hours after storage and one for a viability check after 5 years, the last (2) straw(s) is/are (a) spare in case of leakage after sealing.

3 Revival and viability check

3.1 Organisms are revived immediately after storage and after 5 years. For revival, straws are thawed in a waterbath for five min at 30C. (Some species or groups may not survive 30C; for example, Oomycota are thawed at 25C). The straws are rinsed in ethanol 96%, opened with a sterilized pair of scissors and placed on the suitable agar medium. Growth and identity of the 5 to 8 plugs per dish is checked by the specialists and these data are recorded in a logbook and in the stock control system, including eventual remarks. Changes in morphology are also noted in the CBS database.

Literature

Stalpers, J.A., A. de Hoog & IJ.A. Vlug, 1987. Improvement of the straw technique for the preservation of fungi in liquid nitrogen. Mycologia 79: 82-89.


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

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