Characterizing microbiome dynamics - Flow cytometry based workflows from pure cultures to natural communities

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows from sampling to data analysis for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively.
Conclusion:
We present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the sample sets. We elaborate the cytometric analysis and cell sorting with a complex research centered and an application focused bench-top device, and we suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets. Cytometers were calibrated daily in the linear range with 1 μm beads (FluoSpheres F8815 (350/440), lot no.: 69A1-1) and 2 μm beads (FluoSpheres F-8827 (505/515), lot no.: 1717426), both from Molecular Probes (Eugene, Oregon, USA) and in the logarithmic range with 0.5 μm and 1 μm beads (both Fluoresbrite BB (360/407), lot no.: 552744 and 499344, PolyScience, Niles, Illinois, USA) and 0.5 and 1 µm beads were also added to each sample. A biological standard was used as a staining control. Biological and technical replicates were taken and measured to ensure the precise methodically used workflow. Additionally we measured fixation stability to guarantee result validity over time.