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M/1998/3.00 Appendix 5.11



Various materials such as starch, peptone, dextran or skim milk have been used to make pre-dried plugs onto which small volumes of cell suspension are dropped. After drying and sealing under vacuum the cultures are stored below 10C.

In M/1998/3.00 Appendix 5.11.1 a flow chart of the vacuum drying procedure is shown. For documentation of each step of the preservation procedure and viability tests protocol form M/1998/3.00 Appendix 5.08.2 has to be used.


1. Preparation of freeze-dried skim milk

1.1 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). Dry the vials.

1.2 Fill the vials with 0,5 ml of 20 % (w/v) skim milk (Bacto Skim Milk, dehydrated, Difco 0032)1 with or without additional protective agents.

1.3 Plug the vials with cotton wool plugs for dentists (40 mm in length; size no. 12 for 43 x 10 vials, size no. 23 for 44 x 11 vials). The cotton plug is pushed down to about 10 mm above the skim milk.

1.4 Sterilize the vials at 115C for 13 minutes together with a sterilization control indicator (ATI Steam-Clox)4. For effective sterilization it is best to place the vials loosely in metal racks. Several racks, one upon the other, may be used.

1.5 After sterilization the vials are frozen at -20C for at least 2 hours. The milk is then freeze-dried overnight (with large batches the freeze-drying period has to be extended for one or two days) and may be stored at room temperature until use.

1Other skim milk preparations often are not suitable 2 Dental rolls Art. No. 100001, Roeko, D-89122 Langenau

3 Celluron Zahnwatterollen, Paul Hartmann AG, D-89522 Heidenheim 4 Biologische Arbeitsgemeinschaft GmbH,

D-35423 Lich

2. Sterility testing of freeze-dried skim milk preparations

2.1 Under the sterilization conditions applied certain spore forming bacteria, if present in the skim milk preparation, may survive. Therefore, after sterilization the freeze-dried milk has to be tested for sterility. From each batch of freeze-dried milk the plugs of 10 vials are loosened by tapping the vials. The edge of the vials are flamed and allowed to cool down. The milk plugs are tipped into tubes containing 4,5 ml of sterile Trypticase Soy Yeast Extract Broth. If the edge of the vials are not allowed to cool down, the milk plugs will stick to the glass.

2.2 Five of the tubes are incubated at 28-30C, the remaining 5 tubes at 55C for at least 5 days. The latter incubation temperature is for the detection of thermophilic spore-forming bacteria which are often more resistant to heat than mesophilic organisms. Checking for growth should be at daily intervals. For the detection of anaerobes additional tubes may be incubated in anaerobic jars.

2.3 After incubation 1 drop of each tube is transferred to separate areas of a plate with the selected medium (for both incubation temperatures a separate plate should be used). The plates are incubated for several days at the appropriate temperatures. If growth develops from two or more transferred drops the whole milk batch should be discarded. In case where growth develops from a single drop, sterility testing should be repeated using 10 samples.

3. Control form for skim milk batches

For each batch of sterilized and tested skim milk a control form [M/1998/3.00 Appendix 5.11.2] is used, where data on the preparation, sterilization and sterility testing of skim milk batches are entered. All strains preserved with the same milk batch are also entered into the form with their accession no., together with the date of preservation.

4. Labelling of vials

Before adding cells for preservation labels are fixed to the vials showing the accession number of the strain and month and year of preservation.

5. Preparation of outer tubes

5.1 Place a few grains of self indicating silica gel in 15 x 135 mm tubes. The silica gel becomes blue during drying. (If ampoules are not completely sealed under vacuum the silica gel becomes pink during storage.)

5.2 Cover the silica gel with a small amount of cotton.

6. Filling and drying of vials

Before preparing the cell suspension the freeze-dryer or the "Chemie-Hybrid" vacuum pump is switched on.

6.1 To each vial containing predried skim milk 0.025 ml of a bacterial suspension or one drop from a Pasteur pipette is transferred to the top of the skim milk plug.

6.2 The cotton plug of the vials is replaced. The projecting part is cut off.

6.3 The vials are transferred to the outer tubes. To protect the cotton from heat the vials are covered with a 1-2 cm layer of glass wool.

6.4 The tubes containing the vials are constricted and attached to the manifold of a freeze-drying machine or a "Chemie-Hybrid" vacuum pump. Close the air admittance valve and apply vacuum.

6.5 Dry for a minimum of 6 hours. Seal off the tubes under a vacuum of at least 0.1 mbar.

6.6 Store the sealed ampoules below 10C.

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