Experiment Overview
| Repository ID: | FR-FCM-ZYW5 | Experiment name: | Characterizing microbiome dynamics - Flow cytometry based workflows from pure cultures to natural communities | MIFlowCyt score: | 44.12% |
| Primary researcher: | Johannes Lambrecht | PI/manager: | Johannes Lambrecht | Uploaded by: | Florian Schattenberg |
| Experiment dates: | 2017-07-01 - 2018-02-23 | Dataset uploaded: | Apr 2018 | Last updated: | Apr 2018 |
| Keywords: | [flow cytometry] [fixation] [DAPI staining] [microbiome] [flowCHIC] [flowCyBar] | Manuscripts: | [29084543] [PMC5663091] |
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| Organizations: |
Helmholtz-Centre for Environmental Research - UFZ, Env. microbiology, Leipzig, (Germany)
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| Purpose: | 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. | ||||
| Comments: | None | ||||
| Funding: | This work was funded by the Fachagentur Nachwachsende Rohstoffe e. V. (FNR, Project Biogas-Fingerprint Nr. 22008313) on behalf of the German Federal Ministry for Food and Agriculture (BMEL), the Central Innovation Programme for SMEs (ZIM) of the federal ministry of economic affairs and energy (BMWi) (INAR-ABOS, 16KN043222), the Deutsche Bundesstiftung Umwelt (DBU, Project On-demand Produktion von Phosphatdünger aus Reststoffen von Brauerei und Kläranlage 33960/01-32), the German Federal Ministry for Education and Research (BMBF) (FZK 03XP0041G), and the Helmholtz Association’s Program-oriented funding (POF III R31 topic 3 Bioenergy). | ||||
| Quality control: | 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. | ||||
