I have tried to preserve M. tuberculosis in glycerol and kept at -20C. On subculturing on LJ the recovery has not been good. Also strains die on repeated subculturing on LJ. Can anybody give a practical method of preserving this organism for future use?
We use glycerol at -70 C and have no problems regrowing strains. We do freeze rather large inocula, just to be on the safe side.
I normally grow cells on 7H10 OADC agar until a sufficient lawn of bacteria is present (3 weeks). I then make a mcfarland 3 suspension of the cells in 1 x PBS + 0.05% v/v tween 80. You can also use Sauton or 7H9 OADC media. I then store the suspension in cryovials with glycerol (20% v/v glycerol). I make 1 ml aliquots and vortex them. I store at - 80 degrees. Before use, I thaw the cryovials at room temperature and swab onto 7H10 OADC agar. I get great growth after 2 weeks. This method of storage should be stable for years.
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