期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 137, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/58033
关键词
This Month in JoVE; Issue 137; Flow cytometry; single cell analytics; microbial community dynamics; microbiome; sample preparation flow cytometry; bioprocess control; monitoring cell dynamics; cytometric data analysis; flowCHIC; flowCyBar
资金
- Fachagentur Nachwachsende Rohstoffe e.V. (FNR) [22008313]
- Central Innovation Programme for SMEs (ZIM) of the federal ministry of economic affairs and energy (BMWi) (INAR-ABOS) [16KN043222]
- Deutsche Bundesstiftung Umwelt (DBU, Project On-demand Produktion von Phosphatdunger aus Reststoffen von Brauerei und Klaranlage) [33960/01-32]
- German Federal Ministry for Education and Research (BMBF) [FZK 03XP0041G]
- Helmholtz Association's Program-oriented funding (POF III R31 topic 3 Bioenergy)
The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, 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 respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.
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