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Bacterial mock communities as standards for reproducible cytometric microbiome analysis


Flow cytometry has recently established itself as a tool to track short-term dynamics in microbial community assembly and link those dynamics with ecological parameters. However, instrumental configurations of commercial cytometers and variability introduced through differential handling of the cells and instruments frequently cause data set variability at the single-cell level. This is especially pronounced with microorganisms, which are in the lower range of optical resolution. Although alignment beads are valuable to generally minimize instrumental noise and align overall machine settings, an artificial microbial cytometric mock community (mCMC) is mandatory for validating lab workflows and enabling comparison of data between experiments, thus representing a necessary reference standard for the reproducible cytometric characterization of microbial communities, especially in long-term studies. In this study, the mock community consisted of two Gram-positive and two Gram-negative bacterial strains, which can be assembled with respective subsets of cells, including spores, in any selected ratio or concentration. The preparation of the four strains takes a maximum of 5 d, and the stains are storable with either PFA/ethanol fixation at –20 °C or drying at 4 °C for at least 6 months. Starting from this stock, an mCMC can be assembled within 1 h. Fluorescence staining methods are presented and representatively applied with two high-resolution cell sorters and three benchtop flow cytometers. Benchmarked data sets allow the use of bioinformatic evaluation procedures to decode community behavior or convey qualified cell sorting decisions for subsequent high-resolution sequencing or proteomic routines.

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Fig. 1: Workflows for the preparation of the liquid, plate and dried mCMCs.
Fig. 2: Construction of the liquid mCMC and establishment of the gate template.
Fig. 3: Construction of the plate mCMC and establishment of the gate template.
Fig. 4: Dependence of the mCMC mock community patterns on instrumental setup and instrument type.
Fig. 5: Construction of the liquid mCMC and plate mCMC at varying proportions.
Fig. 6: Influence of changes in sample treatment on the position of cells in the gate template of the plate mCMC and of microbial communities harvested from activated sludge, digester fluid and centrate.

Data availability

The cytometric data sets generated during the current study are available under FlowRepository:, no. FR-FCM-Z2CJ. The Illumina MiSeq data sets generated during the current study are available under BioProject:, accession no. PRJNA541369.

Code and software availability

All links to the R scripts used in the current study are listed in the Equipment section.

R package flowCybar:

R package flowCHIC:

R script for the evaluation of the microscopic pictures (, developed by C. Lepleux).


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This work was funded by the Central Innovation Programme for SMEs (ZIM) of the Federal Ministry of Economic Affairs and Energy Germany (BMWi; INAR-ABOS,16KN043222), the European Regional Development Funds (EFRE—Europe Funds Saxony, grant 100192205) and the Helmholtz Association within RP Renewable Energies. We thank C. Lepleux for developing the script for evaluation of the microscopic data ( and Z. Liu for creating Fig. 6 and Supplementary Methods 3.

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Authors and Affiliations



N.C., T.H. and F.S. performed the experiments and prepared the samples. N.C., T.H., F.-M.K. and F.S. measured the samples. N.C. and T.H. performed the data analysis. N.C., F.-M.K., J.O. and S.M. wrote the manuscript. T.H. and S.M. conceived the ideas and designed the study. S.M. supervised the work. All authors contributed critically to the drafts and gave final approval for publication.

Corresponding author

Correspondence to Susann Müller.

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The authors declare no competing interests.

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Key references using this protocol

Ludwig, J., Höner zu Siederdissen, C., Liu Z, Stadler, P.F. & Müller, S. BMC Bioinformatics 20, 643 (2019):

Liu, Z. & Müller, S. Cytom. Part A (2020):

Protocol Extension

This protocol is an extension to Nat. Protoc. 8, 190–202 (2013):

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Supplementary Information

Supplementary Figs. 1–4, Supplementary Methods 1–3 and Supplementary Tables 1–6.

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Cichocki, N., Hübschmann, T., Schattenberg, F. et al. Bacterial mock communities as standards for reproducible cytometric microbiome analysis. Nat Protoc 15, 2788–2812 (2020).

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