Filling of ampoules with cell suspensions
The ready ampoules (containing a thin disc of carrier material) are equilibrated at 2†-25’ C for few minutes. To each ampoule about 0,025 ml (1 drop with a Pasteur pipette) of cell suspension are added aseptically onto the thin-disc with care so as not to touch the sides of the ampoules. The ampoules are quickly placed in metallic lids and transferred into a metallic jar maintained at 20 ’C in a water bath (see Fig.1 A).
After 20 to 30 minutes of equilibration these are subjected to drying under vacuum. For strains very sensitive to oxygen, the ready ampoules are first placed for few hours in anaerobic Bio-bags (Type A, Marion Scientific Corporation, Kansas City, Ml) or Anaerocult P bags (E. Merck, Postfach 4119, D-6100 Darmstadt). These are carefully taken out one by one and after the addition of reduced cell suspension the ampoules are immediately transferred again to such fresh anaerobic bags, sealed and kept for about one hour at room temperature for equilibration and removal of oxygen. Thereafter, these are subjected to drying under vacuum.