Department of Mechanical Engineering, University of Washington, Seattle, WA.
Successful long-term preservation of Mycobacterium tuberculosis (MTB) cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, M. bovis Bacillus Calmette-Guérin (BCG) and M. tuberculosis H37Ra were used as models of MTB complex strains to study cryopreservation of MTB complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely phosphate buffer saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. Efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight live/dead staining. In all sample matrices examined, the microbiological culture results showed that cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much greater MTB complex recovery rates than rapid cooling (direct immersion into liquid nitrogen) (p<0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9+glycerol), there was no significant differential effect on viability (p=0.06∼0.87). Preincubation of thawed MTB complex cells in 7H9 broth for 20 hours before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (p=0.14∼0.71). The BacLight live/dead staining kit, based on SYTO9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live" cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to cell wall and/or membrane, but instead cause intracellular injury, which leads to the loss of cell viability.