LABORATORY PROCEDURES FOR MICROORGANISMS
M/1998/3.00 Appendix 5.10
PRESERVATION OF BACTERIA BY CENTRIFUGAL FREEZE-DRYING
In the centrifugal freeze-drying process, the suspension is frozen by loss of water by evaporation in vacuum. To avoid frothing of the suspension due to removal of dissolved gases before freezing is complete, the suspension is centrifuged during the initial stages of drying.
For documentation of all steps of the preservation procedure protocol formM/1998/ 3.00 Appendix 5.08.2 has to be used. A flow chart of the process is shown in M/1998 /3.00 Appendix 5.10.1.
1 Preparation of vials
Glass vials (43 x 10 mm, flat bottom) are washed with a detergent, first rinsed in tap water and then in purified water (ion exchange). The vials are plugged with cotton wool plugs for dentists, size no.1 (40 mm in length)1 and sterilized at 121°C for 20 minutes together with a sterilization control indicator (ATI Steam-Clox2). The vials are stored at room temperature. Before use the vials are labelled with the appropriate collection number3 and the month and year of preservation.
Outer glass tubes measuring about 15 x 135 mm are prepared by placing a few pieces of self indicating silica gel into the tubes which are covered with a small amount of cotton wool. During storage of the final ampoules the silica gel will change from blue to red where ampoules are not completely sealed.
1 Dental rolls no. 100001, Roeko, D-89122 Langenau 2 Biologische Arbeitsgemeinschaft GmbH, D-35423 Lich
3in certain cases the collection number may be replaced by the strain designation
2 Suspending medium
Chopped Meat Medium (without the addition of meat) is supplemented with 12 % sucrose. The medium is prepared and prereduced according to the description given in M/1997/2.04 Appendix 2, dispensed in 5 ml amounts into Hungate tubes while these are flushed with oxygen-free nitrogen and sterilized by autoclaving for 20 min at 121°C together with a sterilization control indicator. The suspension medium may be stored at room temperature for several months.
The suspending medium is used both for the preservation of aerobic and anaerobic bacteria. In case of anaerobes, however, tubes with 5 ml of the suspension medium are supplemented with 0.3 ml of a FeS suspension (20 mg FeS ml-1) which is added just before use. The ferrous sulphide is prepared according to Brock and O'Dea (Appl. Environ. Microbiol. 33, 254-256, 1977). Inclusion of ferrous sulphide provides good protection of the cells against oxygen and allows distribution of the suspension under aerobic conditions.
3 Preparation of cultures
Cultures are grown aerobically or anaerobically in culture media as listed in the Catalogue of Strains or in the Accession Form and are usually harvested during active growth. Sporeformers may require special harvesting times.
3.1 Aerobic bacteria are grown on agar slants or in liquid culture and are harvested by washing off or centrifugation.
3.2 Anaerobic bacteria and archaea may be grown in screw-capped bottles, serum bottles, serum tubes (Balch type) or Hungate tubes and have to be transferred under anaerobic gas to centrifuge tubes to collect the cells. Anaerobic bacteria may be grown also in heavy-walled, round-bottomed bottles (c. 70 ml volume) (made by glass blowers, e.g. Ochs GmbH, D-37120 Bovenden-Lenglern) with necks that can be closed with a butyl rubber septum and a screw-cap as with Hungate anaerobe tubes (Bellco Glass Inc., 2047-16125). They fit into a normal laboratory centrifuge and are used both for growing the cells and for centrifugation.
The cultures are centrifuged directly in the unopened bottle, after which the bottle is opened, a gassing cannula inserted and the supernatant aseptically removed as completely as possible using a 20-50 ml hypodermic syringe and a long needle of 2 mm diameter. The gas corresponds to the gas required for preparing the anaerobic medium, except for media under CO2 were N2 + CO2 should be used (otherwise, capillaries may explode when opened!). To the cell pellet, 0.5-1.5 ml of suspending medium is added and cells are suspended by means of a Pasteur pipette, bent and heat-sealed at the tip. Cell suspensions of one or more bottles are collected and transferred to the tube containing the suspending medium which is continuously flushed with oxygen-free N2 gas.
4 Filling vials
The vials, as prepared above, are filled with 0.15 ml of cell suspension. Filling is carried out under aerobic conditions using a calibrated Pasteur pipette or an Eppendorf Multipette 4780 with 2.5 ml Combitip. One sample volume is used to inoculate a fresh culture tube for viability determination.
5 Freezing of suspensions and primary drying
5.1 Close the air-admittance and condenser drain valve of the freeze-drying machine and turn on the refrigerator and the vacuum pump. Allow the condenser (cold trap) to cool down to -40°C to -50°C and allow the pump to warm up for about 30 min.
5.2 Place the vials in the centrifuge head of the freeze drying machine and replace the chamber jar on the baseplate.
5.3 Switch on the centrifuge and, only thereafter, apply vacuum.
5.4 Centrifuge at 760 rpm for 2-3 hours.
5.5 Switch off the centrifuge.
5.6 Continue primary drying until the vacuum has dropped to at least 0.1 mbar. This may be reached after about 1 hour.
Note: Primary drying is incomplete if all the ice has not disappeared or if the vials removed from the chamber are still cold. This material will shrink soon after removal from the drying chamber and should be discarded.
5.7 Switch off the refrigerator and the vacuum pump. Allow air to slowly enter the vacuum chamber via the air-admittance valve. Alternatively, the evacuated system may be flooded with nitrogen gas (recommended especially for anaerobes). The vials are removed from the centrifuge head.
6. Secondary drying
6.1 The projecting parts of the cotton-wool plugs of the vials are cut off. The vials are placed in outer glass tubes containing silica.
6.2 To protect the cotton wool from heat during constriction, the vials are covered with glass wool slightly compressed to a layer 1-2 cm deep. The outer tubes are constricted just above the glass wool.
6.3 When cool the vials are attached to the manifold of the freeze-drying machine for secondary drying at least for 2 hours or overnight.
6.4 At a vacuum of at least 0.1 mbar, the tubes are flame-sealed at the middle of the constriction.
6.5 The ampoules are stored at +8°C or below in the dark.
7. Viability testing
The viability or the colony forming units of the strain are tested before and after the preservation step and, depending upon the strain, at certain intervals during storage. For documentation of the viability protocol formM/1998/3.00 Appendix 5.08.2 is used.
Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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