LABORATORY PROCEDURES FOR MICROORGANISMS
M/1998/3.00 Appendix 5.02
PRESERVATION OF BACTERIA BY FREEZING AND LOW TEMPERATURE STORAGE IN
Polypropylene straws can be used for freezing and storing bacteria in liquid nitrogen. The method is especially useful for the preservation of organisms forming large filaments or mycelium or clumps or when larger amounts of suspensions has to be used for preservation.
For recording the preservation process and viability checks use formM/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.
1 Preparation of straws
1.1 Coloured polypropylene (drinking) straws (from a supermarket) or straws used for the storage of semen at artificial insemination centres (outer diameter 3 mm, MINITÜB GmbH, D-84184 Tiefenbach) are cut into about 3 to 5 cm length.
1.2 On end of a straw is sealed by holding firmly in a pair of unridged forceps 1 mm inwards so that the projecting end is 1 cm from the flame of a fish-tail Bunsen burner. The polypropylene melts almost immediately and forms a strong seal that sets firm within about 10 seconds. Alternatively the straw is sealed by holding it very near to the small flame of a Bunsen burner.
1.3 Several straws are placed in a tube and sterilized at 121°C for 20 min. Different coloured straws may be used for colour-coding strains.
2 Preparation of cultures
2.1 Strains are grown on media and under conditions listed in the catalogue or in the accession form.
2.2 Prepare a heavy suspension of the original culture received or -in case the culture was received in freeze-dried form- from the first subculture in 10% glycerol or 5% dimethylsulphoxide.
3 Filling and freezing of straws
3.1 The sterilized straws are transferred to a petri dish. A single straw is removed with forceps and filled with the cell suspension using a Pasteur pipette. When filling it is necessary to place the end of the Pasteur pipette close to the sealed end of the straw and to fill to within 3 mm of the open end. The filled straw is then sealed at the open end as described above. The inside of the straw at the open end should be kept dry during filling as wet straws do not seal well.
3.2 The closed straws are transferred to a cryotube. Freezing is carried out by placing the cryotube in the upper part of the gas phase of a liquid nitrogen tank for a minimum of 60 min. Thereafter the straws are transferred to a cryotube already fixed to an aluminium cane, which is placed in one of the plastic or aluminium sleeves at a defined position within the LN2 container.
Note: Some bacteria need controlled freezing for good survival. In this case the straws are transferred to a cryotube which is placed in a freezing box (Nalgene). The box is placed for 4 hours in a freezing cabinet of about -75°C. Thereafter the straws are transferred to the liquid nitrogen storage tank.
3.3 For revival a straw is removed from the cryotube and placed immediately in a screw-cap bottle containing water at 30 to 35°C. For disinfection of the outer surface the straw is placed shortly in 70% ethanol. One end of the straw is then cut off using sterile scissors. The contents are removed using a Pasteur pipette and transferred in liquid medium or an agar plate.
Note: The screw-cap bottle (without water) may be precooled over liquid nitrogen to avoid -upon warming- an explosion of a straw not properly closed. After one minute at room temperature water may be added to the straw.
General safety precautions for handling liquid nitrogen (see
Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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