Microfluidic droplets were generated at a flow focusing unit using Novec HFE7500 (3M, Germany) with 0.5% Pico-Surf 1 (Dolomite, UK) as continuous phase. Droplets were generated at 1500 Hz with a volume of 200 pL. One droplet population, which was incubated in one droplet incubator, comprised approximately 9 × 106 droplets. Droplet populations were generated with eight replicates and compared to eight plate sets. One plate set consisted of eight plates with 1:100 dilution of the soil community and seven plates with 1:1000 dilution. The conducted replicates are regarded as biological since independent cultivation experiments were started from one common soil cell extract. The chosen sample size represents a compromise between maximum available droplet incubation units and minimum required replicate number for maximum statistical power.
For each cultivation technique, three positive controls were prepared and incubated along with the soil community samples. After an incubation time of 28 days, microcultures of one droplet population were pooled by fusing the emulsion with 200 µL/mL 1H,1H,2H,2H-perfluoro-1-octanol (Sigma, USA). The upper aqueous phase including the cells was transferred into a new vessel, and the cells were pelleted. From plates, colonies were pooled by suspending the cells in 2 mL 0.9% NaCl solution per plate with a cell scraper.
For each cultivation technique, three positive controls were prepared and incubated along with the soil community samples. After an incubation time of 28 days, microcultures of one droplet population were pooled by fusing the emulsion with 200 µL/mL 1H,1H,2H,2H-perfluoro-1-octanol (Sigma, USA). The upper aqueous phase including the cells was transferred into a new vessel, and the cells were pelleted. From plates, colonies were pooled by suspending the cells in 2 mL 0.9% NaCl solution per plate with a cell scraper.