## 3 Results
### Characteristics of C. albicans Cultured Post Flight  
Many outcome measures can be obtained from experiments conducted to completion on the ISS, yet for reasons of feasibility (especially working with a potential pathogen), as well as sample access and efficient use of crew time, we were interested in determining whether samples could be cultured in microgravity, returned live, and assessed immediately upon return with ground-based procedures. As preliminary evidence from the data obtained on SpX CRS-1 and presented in Figure 3A, it was clear that yeast transitioned to 4 \({}^{\circ}\)C following the growth period in microgravity remained viable and were capable of continued growth. In those studies, viable samples from the Micro-6 payload were scheduled to be returned at 4 \({}^{\circ}\)C, but the unavailability of cold storage resulted in transition to ambient temperature, as illustrated in Figure 2A. The yeast continued to grow during transport as observed in Figure 3A ('Viable Low' and 'Viable High'). In retrospect, it was determined that flight samples were maintained at approximately 5 \({}^{\circ}\)C higher than terrestrial controls for nearly three days, likely contributing to the relative higher growth observed in the flight samples. As a quantitative analysis of post-flight viability, yeast cells were plated to evaluate colony forming units (CFU) relative to that expected based on direct cell counts. As illustrated in Figure 6A, the colony forming potential, and thus cell viability, following the Micro-6 mission was similar between flight and terrestrial controls, regardless of inoculum concentration. Interestingly, when the same analysis was performed on samples in the Micro-14/FPA payload, with transport temperatures maintained at \(<\)6 \({}^{\circ}\)C, terrestrial controls had a rate of viability double that of samples cultured in microgravity (Figure 6B). CFU analysis was also performed on yeast that were cryopreserved (5% glycerol) during the Micro-14/FEP mission. As observed in Figure 6C, the viability of cryopreserved yeast following propagation in microgravity is comparable to that of terrestrial controls. Although cryopreservation is a well-documented mechanism to maintain viability, pre-flight ground-based studies indicated that C. _albicans_ is surprisingly hardy when frozen in the absence of a cryopreservation agent. Non-flight associated, ground-based studies demonstrated that although there is a rapid reduction in viability of cells stored at \(-\)80 \({}^{\circ}\)C, 50% of cells survive when frozen for 50 days, and 30% survive frozen for 100 days when compared to cells stored for the same amount of time in 5% glycerol (unpublished data). Considering this resiliency, cells frozen in microgravity without cryopreservation in Micro-14/FEP (Small FEP bags) were plated to determine viability. Surprisingly, yeast cultured in microgravity were remarkably more susceptible to freeze damage in the absence of a cryopreservation agent than were the terrestrial controls (Figure 6D). Similarly, flight samples frozen without cryopreservation in Micro-14/FPA also had a 5.9- to 10-fold lower viability (as per CFU) than did the terrestrial samples (data not shown). The discrepancy of survival between flight and control samples observed in these studies is intriguing. Due to reduced convection, the samples in microgravity are likely to freeze at a slower rate than terrestrial controls, yet a slower rate of freezing is often associated with improved cell viability [46; 47]. Thus, the freezing rate is not likely to explain the difference in survival between microgravity and terrestrial cells. Indeed, ground-based studies comparing fast and slow freezing rates did not result in differences in yeast viability (data not shown). In addition, since the cells in Figure 6D were frozen for the return trip to Earth, it seems unlikely that re-entry \(g\)-force would have much impact on cell viability.