Glycerol was sterilised by autoclaving for 15 min at 121 C.
A Mr. Frosty freezing container (ThermoScientific) was used to cool samples to 80 C at a rate of 1 C per minute to a final preservation temperature of 80 C.
Preserved cells were then stored at 80 C. Preserved cells were revived at 1 week and 5 weeks post-preservation by thawing in a water bath at 37 C for 20 min. The cells were transferred to clean plastic tubes and centrifuged (1000g for 20 min) to pellet cells.
The cells were washed twice with MBSM to remove residual cryoprotectant.