## 3 Results
### Characteristics of C. albicans Cultured Post Flight
Cumulatively, the diminished post-flight survival of cells cultivated in microgravity and stored either chilled (Figure 6B) or frozen without cryopreservation (Figure 6D) may suggest cellular adaptations that compromise cell wall or plasma membrane integrity. Current studies are underway evaluating changes in ergosterol, the membrane-associated yeast lipid comparable to cholesterol, levels in microgravity. In addition to quantifying colony numbers, macro-architecture of the colonies was also evaluated. We previously demonstrated that colonies initiated from cells cultured in analog conditions, specifically the ground-based HARV bioreactors, demonstrated a complex and relatively unique morphology referred to as highly irregular wrinkled (HIW) [31]. These colonies also consisted of a high percentage of filamentous cells. We were eager to evaluate whether similar macrostructure was produced from cells cultured in microgravity. As illustrated in Figure 7 (panels A-F), the predominating colony morphology was smooth with agar-invading spike-like projections and larger offshoots, referred to as 'feet' projections in the literature [48; 49]. This colony structure did not vary between  
Figure 6: Viability of yeast cells stored chilled or frozen. At the conclusion of the growth period during the mission, cells were chilled to 4 \({}^{\circ}\)C (**A**) Micro-6 and (**B**) Micro-14/FPA. Alternatively, cells were frozen at \(<\)\(-\)80 \({}^{\circ}\)C and transported to Earth at \(<\)\(-\)25 \({}^{\circ}\)C with (**C**) glycerol as a cryopreservation agent in Micro-14/FEP, Large FEP bags or (**D**) without glycerol in Micro-14/FEP, Small FEP bags. Following sample return and hardware de-integration, cells were immediately isolated, counted, diluted, and plated to quantify colony formation. Colonies were counted after 3 days of growth at 30 \({}^{\circ}\)C. Data is represented as the actual number of colonies relative to that expected based on cell counts. Panel A represents samples activated late in the mission (Late) with either the high inoculum (High) or low inoculum (Low), as previously described. Black bars = flight; green bars = terrestrial controls. * \(p<0.03\), **\(p=0.003\)**, flight vs. terrestrial. flight samples (Figure 7A,C,E) and corresponding terrestrial controls (Figure 7B,D,F) in the Micro 6 (Figure 7A,B) or Micro 14/FPA (Figure 7C-E) payloads. The observed morphology was not representative of the fully smooth structure commonly associated with terrestrial cultures of _C. albicans_[50] nor to the HIW colonies observed almost exclusively from cells cultured in the HARV (Figure 7H) [31]. The observed spikey morphology seems to be specific to cells cultured in FPAs, as those grown in the FEP bags (Figure 7G) were predominantly smooth, with some colonies harboring the more traditional 'feet' projections often attributed to elevated CO\({}_{2}\) conditions [48; 49]. Whether cells produced colonies, and how many, was used to evaluate cell viability as previously described and demonstrated in Figure 6. In addition, the size (diameter in mm) of individual colonies was measured as an indirect indicator of cell health and vigor. The diameter was measured on an average of 15 isolated colonies when possible (there were not that many colonies produced from the non-cryopreserved cells from Micro-14/FEP, Figure 6D). As illustrated in Figure 7I, there was an overall trend for cells cultured in flight to give rise to slightly larger colonies. In conjunction with the yeast growth presented in Figure 3, this is another outcome measure that indicates cells may gain a growth advantage upon exposure to microgravity as compared to terrestrial controls. In addition, the growth  
Figure 7: Colony morphology and size evaluated in yeast samples returned live. Following each mission, cells were isolated immediately following hardware de-integration, counted, diluted, and spread on YPD agar plates. Following 3 days of growth at 30 \({}^{\circ}\)C the plates were transferred to 4 \({}^{\circ}\)C pending analysis.