To 2mls of mid-log culture or 1ml of freshly saturated culture add 1 ml(or an equal volume) of glycerol solution, mix gently, then freeze rapidly in liquid nitrogen or on dry ice. Store at -20°C and - 70°C.
Glycerol solution: (Per 100 ml)
65% glycerol (v/v) ............... 65 ml 100% glycerol
0.1M Mg SO4 .......................... 10 ml 1M Mg SO4
0.025M Tris pH 8.0 ................ 2.5 ml 1M Tris
22.5 ml ddH2O
autoclave to sterilize.