2-D clinostat: simulation of microgravity

For the simulation of microgravity, a commercially available fast-rotating 2-D clinostat (UN-KTM2, Advanced Engineering Services, Japan) was used. The sample holder was slightly modified, to hold up to eleven petri-dishes. TBG-medium was poured in germ-counting petri-dishes (Greiner Bio-One GmbH, Germany), which were labelled with a grid on the backside of the plate. The centre of the grid represented the centre of the petri dish and therefore marked the inoculation position of the respective plate. Each plate was inoculated with 10 µl of a fresh PBS-washed overnight culture and air-dried for 2 min. Plates were wrapped with Parafilm to prevent contaminations and mounted in the 2-D clinostat, secured by a lid and four butterfly screws. The clinostat was placed in a standard laboratory incubator at 37 °C. The rotation axis was aligned parallel to the ground with a spirit level. The clinostat was set to 60 rpm, which correspond to ~0.0161 g residual acceleration assuming a final colony diameter of ≤0.8 cm (~r ≤ 0.4 cm). Control cells were placed at a similar position near the clinostat to mimic similar temperature and humidity conditions, however kept at static 1 g control.