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Methods Enzymol. 2013;531:91-107. doi: 10.1016/B978-0-12-407863-5.00005-8.

Quantifying and identifying the active and damaged subsets of indigenous microbial communities.

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FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA.


Flow cytometry and fluorescent dyes represent valuable experimental tools for studying complex microbial communities, enabling the quantification and sorting of cells with distinct levels of activity or damage, and providing information that can be difficult to infer from metagenomic sequencing alone. Despite this potential, these single-cell methods have seldom been applied to the study of host-associated microbial communities. Here, we present our recently developed protocols utilizing four distinct fluorescent dyes that label cells based on nucleic acid content, respiratory activity, and membrane damage. These methods have been successfully applied to study the trillions of microorganisms inhabiting the human gastrointestinal tract (the gut microbiota), in addition to a collection of isolates from five common gut-associated bacterial phyla. By merging these protocols with fluorescence-activated cell sorting and downstream multiplex 16S rRNA gene sequencing, it is possible to rapidly assess the taxonomic composition of each physiological category. These methods provide an initial step toward a robust toolkit allowing a rapid, culture-independent, and comprehensive assessment of the physiology and metabolic activity of host-associated microbial communities.


Cell damage; Community structure; Flow cytometry; Gut microbiota; High-throughput sequencing; Microbial diversity; Single-cell activity

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