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LABORATORY PROCEDURES FOR MICROORGANISMS

Appendix

M/1998/3.00 Appendix 5.03


PRESERVATION OF SPOROACTINOMYCETES BY FREEZING AND LOW TEMPERATURE STORAGE IN PVC STRAWS

INTRODUCTION

Filamentous bacteria like sporoactinomycetes are easily preserved in liquid nitrogen by punching out, with PVC straws, aerial mycelium with spores together with the agar from confluent growth.

For recording the preservation process and viability checks use form M/1998/3.00 Appendix 5.01.1, the quantity of preserved cultures is entered into form M/1998/3.00 Appendix 5.01.2 and the position of straws in liquid nitrogen is recorded in form M/1998/ 3.00 Appendix 5.01.3.

PROCEDURE

1 Preparation of PVC straws: Polyvinylchloride (PVC) straws (125 mm in length, outer diameter 2 mm) are cut to a length of 25 mm. Open at both ends, the straws are sterilized for 15 min at 121C in small tubes.

2 Preparation of cultures: Strains are grown on agar plates according to the instructions given in the Accession Form or the Catalogue of Strains.

The straws are transferred to a sterile Petri dish. They are taken with sterile tweezers and used to punch out aerial mycelium with spores together with the agar from confluent growth. This procedure is repeated until a straw is completely filled. Several straws, still open at both ends, are transferred aseptically to a sterile cryotube which is then closed with a screw cap.

3 Freezing of straws: To obtain a freezing rate that is close to the theoretical optimum of 1 - 10C per minute, the cryotubes containing the straws are placed in the upper part of the gas phase of a liquid nitrogen tank for at least 30 min. Thereafter the cryotubes are fixed to a free position of an aluminium cane which is placed in one of the plastic or aluminium sleeves at a defined position within the LN2 container. The position of the strain is entered into the LN2 Position Book

4 Reactivation of cultures: One straw at a time is removed from the frozen cryotube with the aid of sterile forceps. Unnecessary warming of the cryotube is avoided by using a precooled styrofoam rack. Using a sterile needle, the agar plugs are squeezed out from the straw and are distributed onto plates or slants with culture medium as listed for the respective strain in the Catalogue of Strains or the Accession Form. Incubation is done at an appropriate temperature until growth is visible.

SAFETY PRECAUTIONS

General safety precautions for handling liquid nitrogen (see M/1998/1.06 Appendix 1) have to be followed. Protective clothing (coat, gloves, glasses) should be used while handling the cryotubes.

Liquid nitrogen will enter the cryotubes if not stored in the gas phase only! Before removal of a straw, these cryotubes have to be left in the gas phase of the nitrogen container until all liquid has evaporated from the tube and the cap can be opened without force.


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