Characterizing Microbiome Dynamics - Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

Area of Science:

• Microbiology and Microbial Ecology
• Biotechnology and Bioanalysis

Background:

• Understanding microbial communities is crucial for ecosystems and biotechnology.
• Next-generation sequencing (NGS) is common for microbiome analysis but is resource-intensive and primarily qualitative.
• Flow cytometry (FC) is underutilized in microbiome research despite its potential for quantitative, at-line analysis.

Purpose of the Study:

• To present comprehensive, standardized workflows for flow cytometric microbiome analysis.
• To address the entry barriers related to sample preparation and data analysis in FC microbiome studies.
• To demonstrate the utility of FC for pure cultures, simple, and complex microbial communities.

Main Methods:

• Development of three distinct workflows for sample preparation (sampling, fixation, staining) tailored to different microbial matrices.
• Application of flow cytometric analysis using both research- and application-focused instruments.
• Inclusion of cell sorting procedures and data analysis pipeline recommendations.
• Proposal and application of essential experimental controls.

Main Results:

• Established optimized protocols for sample processing and staining for diverse microbial samples.
• Demonstrated the capability of flow cytometry to provide relative subcommunity abundances and absolute cell numbers.
• Successfully applied the workflows to analyze pure cultures and complex microbial communities in various media.

Conclusions:

• Flow cytometry is a powerful, accessible tool that complements sequencing for in-depth microbiome analysis.
• The presented workflows facilitate the adoption of FC for quantitative microbiome research and applications.
• Standardized FC methods can enhance the resolution and efficiency of microbial community studies.