Abstract
Optical characteristics of individual bacterial cells of natural communities can be measured with flow cytometry (FCM) in high throughput. The resulting data are visualized in cytometric histograms. These histograms represent individual cytometric fingerprints of microbial communities, e.g., at certain time points or microenvironmental conditions. Up to now four tools for analyzing the variation in these cytometric fingerprints are available but have not yet been systematically compared regarding application: Dalmatian Plot, Cytometric Histogram Image Comparison (CHIC), Cytometric Barcoding (CyBar), and FlowFP. In this article these tools were evaluated concerning (i) the required experience of the operator in handling cytometric data sets, (ii) the detection level of changes, (iii) time demand for analysis, and (iv) software requirements. As an illustrative example, FCM was used to characterize the microbial community structure of electroactive microbial biofilms. Their cytometric fingerprints were determined, analyzed with all four tools, and correlated to experimental and functional parameters. The source of inoculum (four different types of wastewater samples) showed the strongest influence on the microbial community structure and biofilm performance while the choice of substrate (acetate or lactate) had no significant effect in the present study. All four evaluation tools were found suitable to monitor structural changes of natural microbial communities. The Dalmatian Plot was shown to be most sensitive to operator impact but nevertheless provided an overview on community shifts. CHIC, CyBar, and FlowFP showed less operator dependence and gave highly resolved information on community structure variation on different detection levels. In conclusion, experimental and productivity parameters correlated with the biofilm structures and practical process integration details were available from cytometric fingerprint analysis.
Highlights
Flow cytometry (FCM) is a high throughput method for analysis of optical characteristics of cells
COMPARISON OF TOOLS FOR MICROBIAL COMMUNITY ANALYSIS The four methods Dalmatian Plot, Cytometric Histogram Image Comparison (CHIC), Cytometric Barcoding (CyBar), and Flow FP are available to follow variations in microbial community structures based on cytometric fingerprinting (Figure 2)
CyBar, FlowFP, and CHIC show highest similarity in the non-metric multidimensional scaling (NMDS) plots, which was supported by procrustes analysis (Table S2), a mathematical procedure to compare ordination results
Summary
Flow cytometry (FCM) is a high throughput method for analysis of optical characteristics of cells. Its main advantages are the fast analysis, high measuring accuracy, and sensitivity on the single cell level. FCM is widely applied for biological analysis, especially in medical routine diagnosis and medical research. The application of FCM for the characterization of microbial cells is less common (Web of Knowledge “flow cytometry” 85,039 hits, “flow cytometry bacteria” 3093 hits, 2014/03/17). The characterization of complex microbial communities by FCM, which is termed cytometric fingerprinting, is still rare. The following article compares and discusses recently published methods for FCM data analyses of complex microbial communities to expand the application of cytometric fingerprinting
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