PRESERVATION OF BACTERIA BY FREEZE-DRYING (1)

LABORATORY PROCEDURES FOR MICROORGANISMS

Appendix

M/1998/3.00 Appendix 5.08

PRESERVATION OF BACTERIA BY FREEZE-DRYING (1)

INTRODUCTION

In the freeze-drying process water is removed from the frozen sample. Bacteria are suspended in a suitable protective medium, frozen and exposed to a vacuum. After drying the bacteria are stored under vacuum in glass vials.

In M/1998/3.00 Appendix 5.08.1 a flow chart of the freeze-drying procedure is shown. For recording each step of the preservation procedure and viability checks protocol form M/1998/3.00 Appendix 5.08.2 has to be used.

PROCEDURE

1. Preparation of vials

Flat bottomed glass vials (43/44 x 11 mm or 43 x 10 mm) are washed with a detergent, then rinsed in tap water and finally in purified water (ion exchange). The vials are plugged (cotton wool plugs for dentists, 40 mm in length; size no. 1¹ for 43 mm x 10 mm vials, size no. 2² for 44 mm x 11 mm vials) and are sterilized at 121°C for 20 minutes together with a sterilization control indicator (ATI Steam-Clox)³. The sterile vials are stored at room temperature. Before use the vials are labelled with the appropriate collection number⁴ and the month and year of preservation.

^{1 Dental rolls Art. No. 100001, Roeko, D-89122 Langenau ²Celluron Zahnwatterollen, Paul Hartmann AG, D-89522 Heidenheim ³Biologische Arbeitsgemeinschaft GmbH, D-35423 Lich ⁴ The number may be replaced by the strain designation}

2. Suspending medium

The suspension media used for freeze-drying are available in the collections. Routinely, 10% skim milk (Difco 0032-17) or 10% skim milk with 5% sodium glutamate may be used.

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 usually harvested during active growth. Sporeformers may require special harvesting times.

Aerobic bacteria are grown on agar slants or plates or in liquid culture and are harvested by washing off with suspension medium or by centrifugation.

Anaerobic bacteria may be grown in screw-capped bottles, serum bottles or tubes (Balch type), Hungate tubes or Bellco Anaerobic Culture Tubes with butyl stoppers (18 x 124 for roll tubes, 25 x 142 for liquid cultures) under anaerobic conditions or on plates in special incubation bags with an oxygen binding system (Merck Anaerocult IS). Cultures are harvested under air by centrifugation in screw- capped tubes or by washing off plates with suspension medium.

4. Filling vials

The harvested culture is mixed with suspension medium. The vials, as prepared above, are filled with 0.2 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. An equal volume is used to inoculate a fresh culture tube for viability determination.

5. Freezing of suspensions and primary drying

5.1 About 30 min before use, 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 Freeze the vials at -20°C for 30 min.

5.3 Transfer the vials to the drying chamber of the freeze-drying machine and apply vacuum. Continue primary freeze-drying overnight. The vacuum has dropped to 0.1 mbar or less.

Note: Primary drying is not complete if not all ice has 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.

5.4 Close the valve connection with the vacuum pump and allow air to enter slowly the drying chamber via the air admittance valve. With anaerobes, the system may be flooded with nitrogen gas.

5.5 Remove the vials from the drying chamber.

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 from the strain, in certain intervals during the storage period. For documentation of viability form M/1998/3.00 Appendix 5.08.2 is used.

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