Cytometric fingerprints: evaluation of new tools for analyzing microbial community dynamics

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.

Neutral mechanisms and niche differentiation in steady‐state insular microbial communities revealed by single cell analysis

“…In addition, to verify the general trends visualized by the cytometric data 44 whole community and sorted 12 gates were analyzed by 16S rRNA gene amplicon sequencing and evaluated both on the class and genus level (Supporting Information S12, data repository https://osf.io/4tkcg/). The second feature is the variable of interest , which is the change in the community structure over time. Here, a fingerprinting method (Koch et al ., , ) was used where groups of cells with similar cell parameters were declared as an entity type (Ovaskainen and Meerson, ). This is synonymous with a subcommunity, or technically, a gate (Supporting Information S5).…”

Individual cell heterogeneity in Predictive Food Microbiology: Challenges in predicting a “noisy” world

“…Protein interactions, Spatiotemporal dynamics (Suhling et al, 2005) (Hutchison and Venter, 2006;Lasken and McLean, 2014;Saliba et al, 2014) PCR qPCR, RT-qPCR, In situ PCR Gene expression, Single cell identification and characterization (Gao et al, 2011;Hodson et al, 1995;Shi et al, 2011) Fluorescence in situ hybridizationImmunofluorescence -Avi et al, 2014;Davey and Kell, 1996;Koch et al, 2014;Shapiro, 2000) …”

Cyt‐Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2018 Conference Workshops

“…A ten‐minute discussion with the audience ensued under the coordination of the workshop facilitators on predefined discussion points. All audio was recorded to enable accurate reporting and a list of participants' names, association, and email addresses were collected. The set‐up and purpose of MCFC assays, in addition to simple cell enumeration, was discussed with a focus on flow cytometric fingerprinting . Additional questions addressed possibilities to increase resolution for MCFC and the inclusion of appropriate experimental controls. With regards to the screening of microbiomes in human environments, a relatively novel application of MCFC , the discussion focused on sample preparation of, for example, fecal slurry and, more generally, MCFC of any type of particulate‐bound microbes or aggregated microbes (e.g., biofilms).…”