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

TitleQuantifying and identifying the active and damaged subsets of indigenous microbial communities.
Publication TypeJournal Article
Year of Publication2013
AuthorsMaurice, CFerrier, Turnbaugh, PJames
JournalMethods Enzymol
Volume531
Pagination91-107
Date Published2013
ISSN1557-7988
KeywordsBacteria, Flow Cytometry, Fluorescent Dyes, Gastrointestinal Tract, High-Throughput Nucleotide Sequencing, Humans, Metagenomics, RNA, Ribosomal, 16S
Abstract

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.

DOI10.1016/B978-0-12-407863-5.00005-8
Alternate JournalMeth. Enzymol.
PubMed ID24060117
Grant ListP50 GM068763 / GM / NIGMS NIH HHS / United States