## 4 Discussion  
As increasing numbers of people spend longer amounts of time in space, it is imperative to understand the physiological adaptations that humans may experience as well as the threats they may encounter. _C. albicans_ is important in its own right as an opportunistic pathogen, but as a eukaryotic organism, it may also provide a simple system that affords insight into how more complex eukaryotes respond to the environment of microgravity. That said, a thorough and robust investigation of biological adaptation to microgravity is complicated for a variety of reasons. Importantly, microgravity cannot be replicated on Earth. Devices that have been developed as simulation bioreactors have not been fully evaluated to determine which aspects of microgravity they simulate, nor the reliability and predictive value they provide. Secondly, opportunities to conduct experiments in true microgravity provide challenges in terms of cost, opportunity, procedures, and outcome measures. Whether utilizing the ISS, suborbital platforms, or free-flyer independent payloads, the conditions, processes, and hardware tend to be unfamiliar and different from  
Figure 8: _C. albicans_ susceptibility to antifungal agents. Micro-6 and Micro-14/FEP were designed to include analyses of the susceptibility of yeast to antifungal agents during cultivation in microgravity. Both payloads included amphoteric B (AmB) at concentrations previously determined to represent a functional range; the Micro-14/FEP payload also contained caspofungin (CAS) within a functional range. (**A**) Micro-6 samples were activated Early in the mission in the presence of 0.12 mg/L and 0.25 mg/L AmB and control conditions (YPD + DMSO), fixed at the end of the growth period, and cell counts performed upon return, (**B**) Untreated Micro-6 cells that were activated Late in the mission and returned viable were analyzed for antifungal sensitivity (AmB) at the conclusion of the mission using CFU analysis following drug exposure. Micro-14/FEP samples were cultured in the presence of (**C**) AmB at 0.12 mg/L, 0.25 mg/L, and 0.5 mg/L AmB and control conditions (YPD + DMSO) or (**D**) CAS at 0.01 mg/L, 0.025 mg/L and 0.1 mg/L CAS and control conditions, frozen on-orbit, and cell counts were completed following the mission. \({}^{*}p<\) 0.05, \({}^{**}p<\) 0.006, Flight vs. Terrestrial (**A,B**); treated vs. sham (**C,D**). those used to establish the foundational studies on which the experimental concepts have been developed. Finally, payload numbers and size tend to be limited, thereby constraining replicate numbers, ability to repeat experiments, and the breadth of experimental variables that can be tested. As such, we feel very fortunate to have flown _C. albicans_ on several missions, allowing for comparison of a subset of outcome measures, across flight opportunities, in different types of hardware, and with reference to results obtained in the HARY bioreactor as a simulation device. When planning flight experiments there are mission-dependent events that will frame the overall timing of experimental procedures and processes, and importantly, the health and wellness of biological samples. This is a feature rarely encountered in normal ground-based experiments and must be thoroughly considered when planning flight payloads. Based on our experience, the time during which samples must be self-sustaining and without active intervention has shortened, but still involves multiple days. This is evidenced in Table 1 when one considers the time from _launch_ to early _sample access_. In SpX CRS-1, this time lapse was 149+ h (6+ days), whereas the shortest lapse was 60 h (2.5 days) during SpX CRS-17. An additional 30-48 h, at minimum, must be added to allow for pre-launch sample preparation, handover procedures, and time required for vehicle packing. Cumulatively and regardless of the experimental design, there is likely to be a multi-day period during which the samples must be stable without crew intervention.