Freeze-drying
The method of freeze-drying was that described previously [17,22]. Two hundred microliter of cultured
cells, homogeneously suspended in a medium containing of 10% skimmed milk and 1% sodium
glutamate was dispensed into each ampoule,immersed in ethanol at 60 C for 2–10 min, and
then connected to a manifold-type freeze-dryer for 4–20 h. The vacuum was lower than 1 Pa and the
ampoules were sealed by a gas burner to maintain the vacuum. The sealed ampoules were stored at
5 C in the dark. For recovery, 0.2 ml sterilized water or physiological saline was added to the sample
in the opened ampoule and mixed well. The suspension
was then serially diluted in sterilized water
and transferred to the medium for each strain.