LABORATORY PROCEDURES FOR MICROORGANISMS
M/1998/3.00 Appendix 5.12
PRESERVATION OF BACTERIA BY LIQUID DRYING (L-DRYING)
Liquid drying (L-drying; Annear, 1958) is a useful alternative method of vacuum-drying for the preservation of bacteria that are particularly sensitive to the initial freezing stage of the normal lyophilization process. The intrinsic feature of this process is that cultures are prevented from freezing; drying occurs direct from the liquid phase.
Use formM/1998/3.00 Appendix 5.08.2 for documentation of the process and viability testing.
1. Plug and sterilize small glass tubes ( 7 x 100 mm). Switch on the vacuum pump and the freezer of a freeze-drying machine and close the valve between the pump and the manifold. Open the air-bleed valve connected between the pump and the manifold.
2. Prepare a dense suspension of bacteria (approx.1010 cells ml-1) in one of the suspension media used for preservation of the strain. With a syringe with a long cannula (1,0 x 120 mm) dispense 0.1 ml of the suspension into each tube taking care not to leave material on the sides of the tube. Replace the cotton wool plugs, cut off the projecting parts and push the plugs down into the tubes (to about 30 mm above the cell suspension) by means of a glass rod of 3 mm diameter.
3. Place a piece of self indicating silica gel on the top of the cotton plug. The silica gel will become blue during the drying process. During storage it turns pink if a tube was not closed under vacuum.
4. Constrict the tubes about 20 mm above the cotton plug with a hot, pointed flame. (The upper end of the tube may become very hot. Therefore fix a piece of a rubber tube to the end of the tube).
5. Attach the tubes vertically to the underside of the horizontal manifold, clamped above a glass tank containing water at about 20°C. The tubes should be immersed in the water to a depth of about 50 mm.
6. Open the valve between the manifold and the pump. Then close the air-bleed valve very slowly until the vacuum is about 50 mbar. De-gas the tubes for 10 min. This procedure removes most of the air from the system without causing violent removal (bubbling) of dissolved air from the suspensions. (Violent de-gassing is undesirable since the culture would be dispersed up the sides of the tubes into the cotton wool plug). After 10 min de-gassing will be complete. The air-bleed valve is closed fully to allow drying to take place. Adherence to this procedure will prevent freezing of the suspension.
7. Depending on the number of tubes attached to the manifold a vacuum of about 10-3 will be reached in about one hour. The tubes are held at this vacuum for 4 hours.
8. The tubes are then flame-sealed under vacuum at the middle of the constriction.
9. Label the ampoules with the collection number of the strain and the month and year of preservation and store the cultures below 10°C.
Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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