Nature Protocols 2020

PROTOCOL EXTENSION
https://doi.org/10.1038/s41596-020-0362-0
Bacterial mock communities as standards for

reproducible cytometric microbiome analysis
Nicolas Cichocki1, Thomas Hübschmann1, Florian Schattenberg1, Frederiek-Maarten Kerckhof 2
,
Jörg Overmann3,4 and Susann Müller 1 ✉
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
1234567890():,;
of cells, including spores, in any selected ratio or concentration. The preparation of the four strains takes a maximum of 5
1234567890():,;
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.
This protocol is an extension to Nat. Protoc. 8, 190–202 (2013): https://doi.org/10.1038/nprot.2012.149
Introduction
Flow cytometry has become an essential technology for the quantitative study of natural or synthetic
microbial communities in ecosystem, biotechnology and health research1. The technique is useful to
uncover the ecology of microbial communities in managed and natural systems and to monitor the
dynamics and evolution of communities. Bioinformatics tools are used to identify the ecological
concepts driving the community assembly processes2,3, to describe the stability properties4 of the
community and to determine what potential interactions might occur between subpopulations of
microorganisms and their environment5. De Vrieze et al.6 highlighted the need for phenotypic
microbial community data in the field of biotechnology, such as methane or carboxylic acid pro-
duction for future automation processes in Industry 4.0. Such applications require absolute com-
parability of the cytometric data over a timeframe of days up to years. Analytical standards are
therefore necessary both to validate lab workflows and to make it possible to compare results from
multiple experiments—experiments that might be done on different days, at different instrument
settings and by different people. Here we describe the design and preparation of an mCMC—a
mixture of different species of bacterial cells that has been fixed and stained—as a suitable standard
for the analysis of microbial communities by flow cytometry.
Development of the protocol

This protocol is an extension to the protocol on the flow cytometric analysis of microbial commu-
nities published earlier7. Flow cytometry is capable of discriminating between subpopulations of pure
cultures or subcommunities of complex microbial communities based on the measurement of cell
1
Department of Environmental Microbiology, Helmholtz Centre for Environmental Research–UFZ, Leipzig, Germany. 2Center for Microbial Ecology and
Technology, Faculty of Bioscience Engineering, Gent University, Gent, Belgium. 3Leibniz Institute DSMZ–German Collection of Microorganisms and
Cell Cultures, Braunschweig, Germany. 4Institut for Microbiology, Braunschweig University of Technology, Braunschweig, Germany.
✉e-mail: susann.mueller@ufz.de
2788 NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot

NATURE PROTOCOLS PROTOCOL EXTENSION
properties, such as cell density (side scatter (SSC), size (forward scatter (FSC)) or the application of
fluorescent markers that label specific cell components1. The scattering properties of bacteria change
as they grow, and their volumes increase or decrease within minutes. Therefore, light scattering works
well for the characterization of the physiological state of bacterial cells8. When combined with
universal dyes, such as DNA dyes, the resulting fluorescence/scatter two-dimensional (2D) plots allow
differentiation between cell types and phenotypes based on scatter behavior, their GC content and
chromosome copy numbers per cell and, hence, can be used to monitor community structure in a fast
and cost-efficient way9.
In the earlier protocol7, the term ‘community structure’ was defined as the number and position of
gates in a 2D plot as well as the number of cells within a gate. This idea is used to track dynamic
variations in the structures of microbial communities by sampling the habitats within short time
intervals; the differences between the community structures can be visualized by comparing the
number of gates and the number of cells in each gate. High-precision bioinformatics and biostatistics
tools are available for creating and evaluating microbial community fingerprints10 or to calculate
diversity indices, such as Hill numbers3, or stability properties, such as resistance and resilience
values4. The quality of values calculated by these tools depends on the quality of the measured data.
Although these tools accurately detect variations between samples, they cannot compensate for
differences resulting from changes to instrument settings and sample handling.
To address this problem, we propose the mCMC for standardization of the flow cytometric
analysis of communities with the aim of improving the resolution and reproducibility of results
obtained, especially for experiments that require measurements of huge sampling cohorts over longer
time periods.
The mCMC standard consists of three or four pure strains, which together have cell populations
that are distributed over the entire 2D plot (ie, are both in the lower and in the upper range of the
logarithmic scales) and which can be separated by gates in a 2D plot. Furthermore, heterogeneity
owing to subpopulations, arising from different cell cycle states or life cycle states, increases the
complexity of the mCMC. The mCMC helps (i) to adjust the position and resolution range of bacteria
in all commercially available flow cytometers, thus improving the resolution of the instruments and
diminishing the background noise; (ii) to identify and reduce the effect of operator-dependent errors
or variations in cell treatment (e.g., by sampling, fixation and staining); and (iii) to set the cells into
the identical position of a 2D plot during long-term experiments and to prevent deviations in
cytometric community patterns, which supports the use of automatic bioinformatics evaluation
pipelines. The mCMC allows for comparison of data between the same devices in different labora-
tories and can serve as a test community for cell sorting and downstream approaches. The mCMC is
an artificial assembly of strains whose proportions can be changed according to required applications
and also for testing new bioinformatics tools.
Application of the method

Several factors can negatively affect a cytometric distribution of a microbial community. The most
common one is background noise, which is caused by different sources. For example, noise can be
caused by inaccurate alignment of the optics and temporal instability of the lasers. Photomultipliers
are often sensitive to laboratory temperature, humidity and air pressure, which results in variations in
signal intensity and an increase or decrease in instrumental noise. The hydrodynamics might vary,
and particles in the sheath and in the tubes can cause backgrounds in the range of signals from
bacterial cells.
To avoid influences on microbial cytometric fingerprints by instrumental settings, standardized
monodisperse beads are routinely used to align flow cytometers, identify false-positive signals in the
background and ensure identical daily machine settings. Frequently, we use mono- or multi-
fluorescent beads with a size between 0.5 and 1 μm.
Although this type of calibration minimizes potential technical bias, beads do not reveal potential
variations in cell handling, such as inadequate or inaccurate cell fixation and staining or cell dis-
ruption due to rough sample handling (e.g., by sonication or extensive washing steps). Each process of
sampling and sample preservation influences the resulting cytometry data and finds its way into
evaluation procedures. Almost all lab operations can contain pitfalls that lead to inaccuracies in the
results and affect the reliability of data or reproducibility of an experiment. Furthermore, in microbial
communities, different cell types can be differentially affected during the sample preparation steps
due to the cell-specific structure. Therefore, their resulting position in the 2D plot and their ratios
NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot 2789

PROTOCOL EXTENSION NATURE PROTOCOLS
might change depending upon the treatment. Even minor variations in sample handling and pre-
paration can result in a potential loss of rare cells and an over-representation of dominant cells, for
instance. Subsequently, bioinformatics evaluation pipelines can produce pointedly distorted results.
Tools for evaluating cytometric patterns, such as flowCHIC, detect small community structure
variation between samples in the range of 0.5% (ref. 11). Any variation introduced by differing flow
cytometer setups on measuring days or varying, (e.g., user-dependent cell handlings) will cause
artificial ambiguities within cytometric microbial community patterns. Therefore, in addition to
monodisperse beads, biological controls should be considered as alignment standards in microbial
flow cytometry.
The mCMC that we propose consists of different phylotypes (Supplementary Table 1), each of
which has different cell scattering and fluorescence characteristics. The respective cell numbers of the
strains are present in a standardized population ranging in proportions between less than 1% up to
80%. The mCMC serves as a qualitative marker for the position of cell types in a 2D plot and as a
quantitative marker for the composition of the community, because the gates have known cell
numbers. Routine and daily use of an mCMC was shown to guarantee comparability of cytometric
microbial community data that were obtained over longer time periods and tested in different setups.
By aligning microbial community patterns using the mCMC, we straightforwardly identified the
dominance and persistence of subsets of cells in microbial communities and the degree to which they
are replaced. We could show that, although all microbial communities were exposed to niche-
differentiating conditions, they assembled into disparate structures. Although the turnover coeffi-
cients were high (>0.6), the nestedness coefficients were complementary low, and intra- and inter-
community ß-diversity values indicated fast community shifts12. Besides using the mCMC to reliably
study the ecological behavior of microbial communities with the help of biostatistics tools, we used it
as a mock community to develop the automated gating tool flowEMMi to establish a high-
throughput, online algorithm for two-channel flow cytometry10. FlowEMMi is a model-based clus-
tering tool based on multivariate Gaussian mixture models with subsampling and foreground/
background separation. The automated cluster analysis requires precise data generation and is
designed to overcome the user-dependent and time-consuming clustering procedures. Therefore, the
mCMC can serve to develop automated gating procedures because its members have fixed positions
in a 2D plot, and cell numbers can be adjusted according to requirements.
Comparison with other methods

The flow cytometric analysis of microorganisms differs from that of eukaryotic cells where cell types
are usually discriminated by specific antibodies labeled with fluorescent or metal markers13–16.
Moreover, most bacteria found in communities taken from natural habitats are not yet cultivable17,18,
and, therefore, (i) it might be difficult or impossible to produce monoclonal antibodies for use as
specific markers for different cell types. In addition, (ii) bacteria have a cell wall that interferes with
the entry of specific fluorescently labeled probes (e.g., FISH probes) to make certain cell types
quantitatively visible. Even if all cell types in a sample were distinguishable by specific fluorescence or
metal markers, (iii) their specific discrimination would be impossible because flow cytometry does not
support the differentiation of more than 100 cell types. In human samples, up to a maximum of 30
cell types can be distinguished using fluorescent markers13, whereas stable heavy metal isotopes can
distinguish up to 40 cell types16. However, the number of bacterial species in a sample from the
environment is usually orders of magnitude higher19.
Next-generation sequencing (NGS) techniques are state of the art to study microbial communities.
NGS techniques provide high-resolution data on the diversity and function of microbial commu-
nities, but the need to standardize the sequencing workflow in the lab became obvious. In recent
years, the idea of routinely integrating a bacterial mock community of known numbers of genotypes
has been developed (e.g., MBARC26 (ref. 20); ATCC MSA-2002, American Type Culture Collection
(ATCC), https://www.lgcstandards-atcc.org/products/all/MSA-2002.aspx; and ZymoBIOMICS, Zymo
Research) for use in various technologies for shotgun metagenomics as well as amplicon 16S ribo-
somal RNA (rRNA) gene sequencing (Illumina; PacBio; and nanopore sequencing, Oxford Nanopore
Technologies). These bacterial sequencing mock communities are now well-established standards as
they allow testing DNA isolation techniques, primer sets and PCR protocols as well as a quality
control of the sequencing setup and resulting data (e.g., contamination or sequencing errors) and
highlight sequencing depth21. Sequencing mock communities also allows comparing data between
experiments and labs. Key methodology parts, such as numbers of cycles, dual indexing, data

acquisition, algorithms, normalization and elimination of chimeras, can be highly improved using a
bacterial sequencing mock community22–24. However, NGS technologies are typically not applied at
the microbial single-cell level, and when they do (ie, after single-cell sorting), they do not function as
high-throughput applications in a cost- and time-efficient manner25,26.
Expertise needed to implement the protocol

Sampling, sample handling and cytometric analysis is straightforward, and the resulting 2D plots can
be readily evaluated using available bioinformatics pipelines. The step in between, namely the
cytometric measurement, requires both high-resolution cytometers and some experience in handling
cytometers and cell sorters. Not all flow cytometers are able to adequately measure bacteria. The
mCMC can therefore be used as a mock community to test the usefulness of flow cytometers for the
analysis of microbial communities. However, most flow cytometry core facilities provide equipment
to measure such samples. Also, because the cells of the mCMC are fixed, there should be no problem
in infecting living eukaryotic cells that are measured with the same instrument in a core facility.
Limitations
The creation of the mCMC requires the cultivation of mock strains. The strains must always be of the
same origin and kept under standardized conditions requiring a minimum of equipment in a
microbiological laboratory, such as a safety bench and cultivation facilities. Even the cultivation itself
must be standardized. Microbial cells typically change their physiological properties during growth,
which can be well documented by flow cytometry. During the exponential growth phase, they
increase in cell size and multiply the DNA content per cell, changing their position in a cytometric 2D
plot (Table 1). These changes can occur rapidly within minutes. Therefore, actively growing cells are
not useful as standards for microbial flow cytometry. Instead, stationary phase cells should be used.
Additionally, the mCMC is assembled from fixed individual strains and is used to standardize the
measurement of fixed microbial communities. If the mCMC should standardize the measurement of
living cell communities, a dye for live cells has to be used to label the fixed mCMC. When measuring
autofluorescent communities, at least the scatter parameters of the fixed mCMC can be used for
standardization. Generally, the preparation of the mCMC requires more time than, for example, the
use of the sequencing mock community MBARC26, which is available as a ready-to-use DNA sample.
However, because all strains of the mCMC are cultured separately, their respective cell proportions
might be mixed according to experimental requirements, which is not the case for the current
standards available for sequencing approaches. In addition, flow cytometers are sold in a fluctuating
market, which is why manufacturers change their manufacturing focus every few years, depending on
which customers they want to acquire. As a result, optics, modes of hydrodynamics, laser types and
power, as well as hardware and software developments, change each year. In particular, laser power is
a cost issue, and the manufacturers are trying to reduce costs by using low-power lasers. Hence, the
mCMC is a valuable standard to test cytometers for their ability to measure events in the size of
bacteria.
Experimental design
Samples
For the mock community, non-pathogenic bacteria were selected, which are readily available and
cultivable. Both Gram-positive and Gram-negative strains with known fully sequenced genomes were
included. Based on these criteria, Stenotrophomonas rhizophila DSM 14405, Escherichia coli DSM
4230, Kocuria rhizophila DSM 348 and Paenibacillus polymyxa DSM 36 were selected (Supple-
mentary Table 1).
Strain cultivation
In this procedure, two types of mCMCs were constructed (Fig. 1). The strains were pre-cultured
separately on agar plates for 72 h and then cultured either on agar plates for an additional 72 h or in
liquid medium as pre-culture and main culture for 24 h each. The liquid-grown strains were used to
assemble the liquid mCMC, which contains the four strains in their early stationary phase (Fig. 2).
The plate-grown strains S. rhizophila, K. rhizophila and P. polymyxa were used to assemble the plate
mCMC, which contains the three strains in their late stationary phase as well as spores from the
P. polymyxa strain (Fig. 3). Spores have a distinct size and DNA content and are therefore a valuable
subpopulation for the cytometric calibration procedure.

Plating
Paenibacillus polymyxa (DSM 36) 1 72 h – 30 °C
Stenotrophomonas rhizophila (DSM 14,405) Liquid
Kocuria rhizophila (DSM 348) OD700 = 0.05
Escherichia coli (DSM 4,230)
Pre-culture Culture
24 h – 30 °C 24 h – 30 °C
Plating
72 h – 30 °C
Plate
OD700 = 0.5
Cytometric measurement
of the mCMC Dried
104
DAPI fluorescence
2 2
103
5 – 14 4 3
(log)
102
mCMC design Staining with Formaldehyde/ethanol
1 fluorescent dye –20 °C
10 Overnight OD700 = 0.04
RT
100
100 101 102 103 104
FSC (log)
Fig. 1 | Workflows for the preparation of the liquid, plate and dried mCMCs. The protocol starts with the reactivation of the four strains S. rhizophila,
E. coli, K. rhizophila and P. polymyxa (received from the DSMZ) on LB agar for 72 h. From there, the strains were cultivated in liquid LB medium for 24 h
and fixed with a PFA/ethanol procedure. Otherwise, cells can also directly be taken from plate after 72 h and fixed using the same procedure or dried.
Next, fixed or dried cells were separately stained with a fluorescent dye overnight at 20 °C. Different strains and proportions of strains were mixed to
create either the plate or liquid mCMCs used for the calibration of the flow cytometric measurement of microbial communities.
Strain fixation and staining

Samples of the strains were harvested and fixed by a formaldehyde/ethanol-containing solution or by
drying. Subsequently, the cellular DNA was stained with DAPI and analyzed cytometrically (Fig. 1).
DAPI excitation requires a UV laser that is less commonly used in commercial flow cytometers.
Therefore, an additional staining approach has been evaluated that requires a standard 488-nm laser
for excitation. SYBR Green I can be excited at this wavelength and is a nucleic acid dye that resulted
in less, but still sufficient, separation of the strains within the mCMC to serve as standard.
Proportions of strains in either liquid or plate mCMCs

After a standardized cultivation, sampling, fixation and staining protocol, each population (and
its subpopulations) appears segregated in a cytometric 2D plot. An mCMC can be designed
with different proportions of strains based on OD(dʎ700nm = 0.5 cm) to ensure that both smaller
(S. rhizophila) and larger (P. polymyxa) cells in the 2D plot can be detected even if their proportions
are very different (e.g., between 97.5% and 0.5%, respectively; Figs. 2 and 3 and Supplementary
Tables 2 and 3).
Gate setting for the liquid mCMC

All four strains showed typical phenotypic distributions during growth, which change rapidly
(Fig. 2b). Therefore, only stationary phase cells (harvested after 24 h) were used to create the liquid
mCMC (Fig. 2b,d). To ensure the position of the cells in a 2D plot, non-overlapping gates were
established for the most abundant subpopulations of each strain: S. rhizophila (L1 and L2), E. coli
(L3 and L4), K. rhizophila (L5 to L7) and P. polymyxa (L8 to L12) (Fig. 2d). Thus, the gates L1 to L12
constitute the gate template for the liquid mCMC (Fig. 2d).
Gate setting for the plate mCMC

Cells of mock strains were taken either from streaked cells on plates or from dried material thereof
(harvested after 72 h; Fig. 1). Correspondingly, gates were set for the most abundant subpopulations
of three strains: S. rhizophila (P1 to P3), K. rhizophila (P4 to P6) and P. polymyxa (P7 to P11). The
gates P1 to P11 thus form the gate template for the plate mCMC (Fig. 3c). E. coli was excluded
because the gates of this strain were overlapping with the gates of P. polymyxa when grown on plates
for 72 h.
The gate template

The gate template is designed for either the liquid or the plate mCMC (Figs. 2d and 3c). The gate
template guarantees the quality of sample handling and the comparison of samples within mea-
surement campaigns. It is the most important part of the daily alignment procedure on a flow

a S. rhizophila E. coli K. rhizophila P. polymyxa
2.5
OD 700 nm
2
1.5
1
0.5
0
0 5 10 15 20
Time (h)
b
0h
2h
4h
104
DAPI fluorescence (log)
103
102 24 h
101
100
100 101 102 103 104
FSC (log)
c d
0.2
NMDS2
104
0.0
Beads 1 µm
–0.2 103
–0.2 –0.1 0.0 Beads 0.5 µm L11
L12
NMDS1 2 L10
10 L4 L8 L9
0.06 L3 L7
5c L2 L6
0.04 101
2a = 5a 7a 7c L1 L5
0.02
NMDS2
5b
0.0 2c = 4a 2b = 3a 7b 100
4c 6a
3b 100 101 102 103 104
–0.02 4b 3c 6c FSC (log)
–0.04 6b
–0 4
2
0
02
04
06
.0
.0
0.
0.
0.
0.
–0
NMDS1
e
L1 L2
L3 L4
L5 L6 L7
104
103
102
101
100 L9 L11 L12

100 101 102 103 104
FSC (log)

Fig. 2 | Construction of the liquid mCMC and establishment of the gate template. a, Growth curves
‘geom_smooth_alpha’ (confidence interval, 0.95) of the strains S. rhizophila, E. coli, K. rhizophila and P. polymyxa (six
biological replicates, respectively). b, 2D plots (FSC versus DAPI fluorescence) of samples of the four mock strains
that were harvested at 0 h, 2 h, 4 h and 24 h and measured by flow cytometry. Each of the strains is represented by
different subpopulations at different times of growth. The occurrence of the number and size of the subpopulations is
strongly strain specific and depends on the state of the cells in the cell cycle. Blue reference beads (0.5-µm and
1.0-µm) were spiked into the samples. c, Visualization of the degree of dissimilarity between technical and biological
replicates in flow cytometric measurements using Bray–Curtis-based non-metric multidimensional scaling of
21 samples (200 iterations). The mCMCs originating from plate (left) and liquid (right) medium were widely
dissimilar in their cytometric structure. Several setups of technical replicates, stored for 2 months at −20 °C, were
tested using the liquid mCMC (zooming): (2a–c) biological triplicates; (3a–c) technical triplicates of cytometric
measurements of the biological replicate 2b; (4a–c) technical triplicates of cytometric measurements of the
biological replicate 2c; (5a–c, 6a–c, 7a–c) triplicates of the staining procedure of the biological replicate 2a. d, Cell
gate and gate template of the liquid mCMC. The 2D plot (right) represents the constructed mock community
(P. polymyxa, S. rhizophila, K. rhizophila and E. coli at a % ratio of 70:2.5:20:7.5, respectively). The cell gate (left)
represents the cell gate excluding beads and noise and the gate template within the cell gate marks the
subpopulations of the mCMC members: S. rhizophila (L1 and L2), E. coli (L3 and L4), K. rhizophila (L5 to L7) and
P. polymyxa (L8 to L12). e, Cell sorting and re-analysis of subpopulations from the liquid mCMC. Selected sorted
subpopulations were re-analyzed, and their pre-sorted position in the gate template was confirmed. First row:
S. rhizophila (L1 and L2); second row: E. coli (L3 and L4); third row: K. rhizophila (L5 to L7); fourth row: P. polymyxa
(L9, L11 and L12; with L12 cells splitting during the sorting and re-measuring procedure).
cytometer before unknown microbial communities are measured. All strains and their major sub-
populations of the respective mCMC must fit into their specific gates. If they do not fit into their
gates, the fixation and staining procedures must be repeated.
Cytometric analysis and cell sorting for downstreaming applications

The mCMC guarantees comparability between samples. For each flow cytometrically measured
sample campaign, the number of gates and the number of cells in the gates are the basic information
needed to quantify the dynamics and behavior of the community7. In addition, subsets of cells at
particular positions in a 2D plot can be sorted out of a fixed position in a 2D plot, even if rare cells or
certain phenotypic states of cells are well below 1%. This possibility might be particularly important
for the meta-profiling of microbial communities, as these rare cells can be separated from all other
cells of a community and thus processed alone, allowing for more in-depth information using -omics
techniques.
Moreover, the physiological states of microbial cells in a community change owing to growth
conditions, which influence their optical properties and chromosome numbers. These states might
influence how often their DNA is amplified after cell sorting, due to changed cell wall structure, the
number of chromosomal copies or the accessibility of primers to DNA27. Therefore, the mCMCs,
containing cells of the same species with different numbers of chromosomes, and, in addition, spores
of the plate mCMC, are valuable test communities for downstream processing of sorted cells.
The different physiologies of cells in gates of both the liquid and plate mCMCs were reflected in
their morphological variations as well. However, the size of a microbial cell is not reflected linearly by
the FSC of a flow cytometer. To illustrate the differences, the cells of the individual gates were sorted
both from the liquid and the plate mCMCs, and the length and width of the cells were determined
microscopically (Fig. 3d,e). Usually, the cytometric FSC is dependent on the cell size, as shown for the
gates L1 to L7 and P1 to P6, respectively. However, spores (P11) caused the highest possible FSC
signal from all subpopulations on a logarithmic scale but showed a microscopically measured mean
size of only 2.03 ± 0.18 µm compared to exponentially growing L12 cells of P. polymyxa, which had
an average size of 6.36 ± 0.79 µm. The theoretical background is provided by the Mie theory28, which
suggests that single-particle scattering causes nonlinear size-dependent signal intensities of events in
the size range of microbial cells when addressed with forward scattered, bent light at small angles
(2–30° for the BD Influx v7 Cell Sorter).
Controls
The individual strains of the mCMC show specific stationary phase patterns (FSC versus DNA) in a
2D plot. In our experience, the fixed stationary phase cells are stable for at least 6 months. Afterwards,
a new stock of strains should be created according to the described procedure. The stationary phase
patterns for the liquid mCMC (harvested after 24 h) and for the plate mCMC (harvested after 72 h) of
the follow-up batches should show the same distributions. If patterns are different, we recommend

a
S. rhizophila
P1 P2 P3
K. rhizophila
P4 P5 P6
P. polymyxa
P7 P8 P9
104
103
P. polymyxa
102
101
P10 P11
100
100 101 102 103 104
FSC (log)
b P. polymyxa c 104
P7 Beads 1 µm
103
P8 Beads 0.5 µm
2 P7 P9 P10
10
P8 P11
P9
P6
P3
101 P1 P5
P10 P2 P4
P11 100
100 101 102 103 104
FSC (log)
10 µm
d Liquid Plate Liquid Plate
8 2
6 1.5
Length (µm)
Width (µm)
4 1
2 0.5
L1 L2 L3 L4 L5 L6 L7 L9L11 A B C P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 L1 L2 L3 L4 L5 L6 L7 L9 L11 A B C P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11

L12 L12

Fig. 3 | Construction of the plate mCMC and establishment of the gate template. a, Cell sorting and re-analysis of
subpopulations from the plate mCMC. Selected sorted subpopulations were re-analyzed, and their pre-sorted
position in the gate template was confirmed. First row: S. rhizophila (P1 to P3); second row: K. rhizophila (P4 to P6);
third row: P. polymyxa (P7 to P9); fourth row: P. polymyxa (P10 and P11). Blue reference beads (0.5-µm and 1.0-µm)
were spiked into the samples. b, Exemplary phase contrast microscopic pictures of cells sorted from gates of P.
polymyxa (P7 to P11). c, Cell gate and gate template of the plate mCMC. The 2D plot (right) represents the
constructed mock community (P. polymyxa, S. rhizophila and K. rhizophila at a % ratio of 80:1:19, respectively). The cell
gate (left) represents the cell gate excluding beads and noise, and the gate template within the cell gate marks the
subpopulations of the mCMC members: S. rhizophila (P1 to P3), K. rhizophila (P4 to P6) and P. polymyxa (P7 to P11).
d,e, Microscopic analysis of sorted cells in gates L1 to L7, L9, L11, L12 and P1 to P11 of both the liquid and plate
mCMCs: S. rhizophila (tan), K. rhizophila (blue), E. coli (green) and P. polymyxa (violet). Cell length (d) and width (e)
of ~500 sorted cells, respectively, were measured. The data were distributed on the graphs with the marks ‘jittered’
and ‘boxplot overlay’. Cells from L12 were separately represented in three groups (L12 A–C) owing to apparent
diversity of morphotypes.
measuring individual growth phases of the individual strains from time to time to ensure that
each strain still exhibits the expected growth behavior (Fig. 2b). The purity of the strains can be
tested, for example, by Sanger or Illumina MiSeq sequencing techniques (see ‘Procedure’, Step 1(C)).
The different strains or subgroups of cells can also be sorted and sequenced (see ‘Procedure’, Step 14,
Box 2). If patterns do not fit or strains are not pure, we recommend purchasing new strains.
Experimental replication
In this protocol, we suggest workflows for two different mCMCs, and we propose two different
staining procedures. DAPI stains cell chromosomes and is excited by UV light, whereas SYBR Green I
stains nucleic acids and is excited at 488 nm. Similarly to the obligatory implementation of, for
example, the MBARC26 in each sequencing approach, we recommend including an mCMC standard
every day when samples are analyzed. The mCMC should be handled and stained identically to the
user’s method for the samples of an experimental campaign. For example, samples from different
environments (e.g., from fresh water or from the human intestine) might require different sample
preparations for washing and sonicating, the amount of dye or the duration of fixation and staining
procedures. The identical treatment of the mCMC places the mock in a position of the 2D plot
corresponding to the natural community being studied and immediately reveals differences in user-
dependent cell treatments. The resulting resolution of the mCMC should be similar to that shown in
this protocol. If the resolution of the mCMC pattern is worse, the user’s fixation and staining
procedures should be optimized until the resolution fits.
Also, we have successfully demonstrated the stable resolution of the mCMC’s FSC signal at a
wavelength of 488 nm for laser powers ranging from 400 mW to 50 mW, even if the gain of
the photomultiplier tubes (PMTs) was increased to map the mCMC in the original gate template
(Fig. 4a). However, if other devices with different equipment are implemented while sampling
campaigns are running, the community pattern can change dramatically. We measured the mCMC
with a MoFlo Legacy Cell Sorter, another high-throughput flow cytometer (Fig. 4b) similar to the BD
Influx v7 Cell Sorter but different in some optical path characteristics. We have found that the pattern
of the mCMC deviates from the known pattern. Both devices easily recognize beads of 0.5 µm and
1 µm, albeit at quite different positions in the 2D plot. The positions of the strains within the patterns
of the plate and liquid mCMCs were similarly different, although their resolution remained high and
stable. Because this depends on the laser, the PMTs and the optical design of the device, the mCMC
gate template must be separately and newly created for each cytometer before microbial community
data can be reliably evaluated.
Commercial benchtop flow cytometers are frequently not equipped with an expensive UV laser.
We tested whether the mCMC can also serve as a standard for analyzing microbial communities on
these instruments. All devices were equipped with a 488-nm laser, which is commonly used to
measure cell scattering. The laser also excites SYBR Green I. The use of SYBR Green I does not
change the scatter behavior of the bacterial cells (Supplementary Fig. 1). However, we expected that
the overall resolution of the mCMC bacterial subgroups in the fluorescence channel will be lower. The
four and three pure strains of both the liquid and the plate mCMCs were clearly separated with the
BD Influx v7 Cell Sorter, confirming that, with SYBR Green I, also high-resolved community patterns
can be created on research instruments. Lower laser power (from 400 mW to 50 mW) did not reduce
the resolution of these patterns (Supplementary Fig. 1). However, benchtop devices, such as the
analyzers CyFlowSpace and BD Accuri C6+, represent patterns not only at altered positions in the

a 400 mW 200 mW 100 mW 50 mW 50 mW+
104

103
102
101
100
100 101 102 103 104
FSC (log)
b BD influx MoFlo
Liquid
104
103
Plate
102
101
100
100 101 102 103 104
FSC (log)
Fig. 4 | Dependence of the mCMC mock community patterns on instrumental setup and instrument type. a, Liquid mCMC analyzed using a 355-nm
(100-mW) laser for DAPI fluorescence and a 488-nm laser at laser powers 400 mW, 200 mW, 100 mW and 50 mW. The gain values of the
FSC-PMT were increased from 34.15 to 46 for the 50-mW+ 2D plot. b, mCMC liquid (upper row) and plate (lower row) analyzed by cytometers
BD Influx v7 Cell Sorter and MoFlo Legacy Cell Sorter. The cell gates and respective gate templates were set, and 0.5-µm and 1.0-µm blue reference
beads were spiked into the samples.
2D plot but also at lower resolutions. Nonetheless, all benchtop devices resolved subsets of the mock
populations (four to nine for the liquid mCMC and three to ten for the plate mCMC), and spores
(marked by arrows) were detected in all plate mCMCs (Supplementary Fig. 1). These data show that
the mCMC is a good standard for testing the degree of resolution of flow cytometers. Benchtop
devices with lower laser power down to 50 mW (both UV and 488-nm laser) are suitable for
microbial community analysis, albeit with lower resolution. They also show that it is not possible to
compare the same data measured with different instruments.
Additionally, the mCMC can be used to verify the sorting procedure. For microbial communities,
sorting is mainly performed to get information on diversity (or specific potential functions)
of subsamples (16S rRNA gene or other specific gene amplicon sequencing applications) or on
single-cell genomes or submetagenomes29. In addition, for functional relationships between species,
also subpopulation or subcommunity proteomes are of high interest30. For all these applications, a
sort mode should be chosen for cell sorting that ensures the highest possible purity (99% for the BD
Influx v7 Cell Sorter) of sorted subsets of cells. Sorting subsets of cells out of the mCMC and their
successful re-analysis and re-visualization within the identical gate is a perfect control of instrument
and sample handling (Fig. 2e). Furthermore, we used the plate mCMC to sort gate-specific cells on
glass slides to be visualized by microscopy. The cells of gates P7 to P11 (P. polymyxa) clearly represent
different morphotypes of the identical clonal strain, with P10 and P11 revealing different types of
spores (Fig. 3b).

Materials
Biological materials
●
K. rhizophila DSM 348 (German Collection of Microorganisms and Cell Cultures (DSMZ); Leibniz
Institute)
● P. polymyxa DSM 36 (DSMZ; Leibniz Institute)
● S. rhizophila DSM 14405 (DSMZ; Leibniz Institute)
● E. coli DSM 4230 (DSMZ; Leibniz Institute)
Reagents
●
Lysogeny broth medium according to Lennox (LB medium; Carl Roth)
● Glycerol (Carl Roth, CAS no. 56-81-5)
● Reference beads blue 1-µm BB Fluoresbrite Microspheres (Polysciences, cat. no. 17458)
● Reference beads blue 0.5-µm BB Fluoresbrite Microspheres (Polysciences, cat. no. 18339)
● Reference beads yellow-green 1-µm FluoSpheres (Thermo Fisher Scientific, cat. no. F-13081, lot. no.
03B2-1)
● Reference beads yellow-green 0.5-µm FluoSpheres (Thermo Fisher Scientific, cat. no. F-8813, lot. no.
69B3-1)
● Laser alignment beads blue 1-µm FluoSpheres (Thermo Fisher Scientific, cat. no. F-8815)
● Laser alignment beads yellow-green 2-µm FluoSpheres (Thermo Fisher Scientific, cat. no. F-8827)
●
Laser alignment BD FACSuite CS&T research beads (BD, cat. no. 650621)
●
Laser alignment BD CS&T RUO beads (BD, cat. no. 661414)
● Bidistilled water
● Milli-Q water
● Ethanol (Sigma-Aldrich, CAS no. 64-17-5)
● Paraformaldehyde (PFA; Sigma-Aldrich, CAS no. 30525-89-4) ! CAUTION Preparation of the PFA solution
causes noxious fumes. Work under the fume cupboard and wear gloves when handling the chemical.
● Sodium phosphate dibasic (Na HPO ; Sigma-Aldrich, CAS no. 7558-79-4)
2 4
● Sodium phosphate monobasic (NaH PO ; Sigma-Aldrich, CAS no. 7558-80-7)
2 4
● Sodium chloride (NaCl; Sigma-Aldrich, CAS no. 7647-14-5)
● Potassium phosphate monobasic (KH PO ; Sigma-Aldrich, CAS no. 7778-77-0)

2 4
● Potassium chloride (KCl, Sigma-Aldrich, CAS no. 7447-40-7)
●
Magnesium chloride solution (MgCl2, New England Biolabs, product no. B9021S)
● Tris (Thermo Fisher Scientific, cat. no. 17926)
● EDTA (Thermo Fisher Scientific, cat. no. 17892)
● Agarose for gel electrophoresis (Sigma-Aldrich, CAS no. 9012-36-6)
● Difco gel (BD Difco 1.8% agar, BD, CAS no. 9002-18-0)
● Immersion oil Type-F (Olympus)
● Citric acid (Sigma-Aldrich, CAS no. 77-92-9)
● Tween-20 (Sigma Aldrich, CAS no. 9005-64-5)
● DAPI dihydrochloride (Sigma-Aldrich, CAS no. 28718-90-3) ! CAUTION DAPI is toxic. Wear gloves when
handling the solution.

● SYBR Green I (Thermo Fisher Scientific, CAS no. 163795-75-3) ! CAUTION SYBR Green I is toxic. Wear
gloves when handling the solution.

● Dimethylsulfoxide (DMSO; Merck, CAS no. 67-68-5)
● BD FACSFlow buffer (BD, cat. no. 342003)
CRITICAL Always re-suspend the

c
● Chelex 100 sodium form (Sigma-Aldrich, CAS no. 11139-85-8)
powder thoroughly before use. Do not allow Chelex powder to come into contact with metal.
● Gel loading dye (Qiagen, cat. no. 239901)
● Molecular marker (Qiagen, cat. no. 239045)
● Ethidium bromide (Sigma-Aldrich, CAS no. 1239-45-8) ! CAUTION Ethidium bromide is toxic. Wear
gloves when handling the solution. You can also use alternative fluorescent nucleic acid stains, which are
less toxic.
● Molecular-grade water (Sigma-Aldrich, CAS no. 7732-18-5)
●
Nuclease-free water (Qiagen, cat. no. 129115)
● PCR for DNA quality testing
Forward 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ (Eurofins Scientific)

Reverse 1492R 5′-TACGGYTACCTTGTTACGACTT-3′ (Eurofins Scientific)

●
PCR 1 for Sanger and Illumina MiSeq amplicon sequencing
PCR1: Primer 1 Forward Pro341F 5′-CCTACGGGNBGCASCAG-3′ (Eurofins Scientific)
PCR1: Primer 2 Reverse Pro805R 5′-GACTACNVGGGTATCTAATCC-3′ (Eurofins Scientific)
● PCR 2 for Illumina MiSeq amplicon sequencing
PCR2: Six-nucleotides-barcoded mother primers (Eurofins Scientific)

● dNTP (Promega)
● Phusion GC solution (New England Biolabs, cat. no. B0519S)
● Phusion High-Fidelity Polymerase (New England Biolabs, cat. no. M0530S)
● Agencourt AMPure beads XP-Kit (Beckman Coulter, cat. no. A63880)
● dsDNA HS Assay Kit (Thermo Fisher Scientific, cat. no. Q32851)
● Ethanol 70% in bidistilled H O (vol/vol)

2
●
TAE (5×; see ‘Reagent setup’)
−1
● Ethidium bromide staining solution (0.5 µg ml final concentration in bidistilled water)
Equipment
● Cryotubes (Sarstedt)
● Glass tubes (Duran, 10 ml, treated 30 min in 2% Hellmanex III (Hellma Analytics))
● Microcentrifuge tubes (0.2, 1.5 and 2 ml; Sigma-Aldrich)
● Cell Trics (50 µm; Sysmex-Partec)
● Ultraspec III (Amersham Biosciences Europe)
●
Ultrasonic bath DT 100 Sonorex Digitec (Bandelin)
●
Micro-Cenvac NB-503CIR (N-Biotech)
● Centrifuge for glass tubes (Eppendorf centrifuge 5804R, Eppendorf)
● Microcentrifuge (Heraeus Fresco 21 Centrifuge, Thermo Fisher Scientific)
● Vortex (VF2, Janke Kunkel IKA Labortechnik)
● Microwave (Severin)
● BD Influx v7 Cell Sorter (BD; see ‘Equipment setup’ section)
● MoFlo Legacy Cell Sorter (Beckman Coulter; see ‘Equipment setup’)
● CyFlowSpace analyzer (Sysmex-Partec, see ‘Equipment setup’ section)
● BD FACSVerse analyzer (BD, see ‘Equipment setup’ section)
● BD Accuri C6+ analyzer (BD, see ‘Equipment setup’ section)
●
AxioScope A1 fluorescence microscope (Carl Zeiss Microscopy)
●
Zeiss Axiocam MRm camera (Carl Zeiss Microscopy)
● Objective plan-apochromat 100×/1.40 Oil DIC M27 (Carl Zeiss Microscopy)
● Thermomixer (Eppendorf)
● PTC-200 Thermal Cycler (MJ Research)
● PCR machine (S1000 Thermal cycler, Biorad)
● Magnetic separation rack (magnetic Stand-96, Thermo Fisher Scientific)
● DNA quantifier (Qubit 3.0, Thermo Fisher Scientific, cat. no. Q33216)
● Sanger (Eurofins Scientific)
● Illumina MiSeq (Illumina Inc.)
Software
● Silva SSU / LSU database v132 (https://www.arb-silva.de/)
● Image-Insight Pro 9.1 (Media Cybernetics)
● FlowRepository: https://flowrepository.org/, no. FR-FCM-Z2CJ
● FlowJo 10.0.8.r1 (FlowJo, Tree Star / BD)
● Mothur 1.41 (ref.

31
)
● UCHIME
32
● NCBI database (National Library of Medicine, National Center for Biotechnology Information; cited
November 25, 2019). Available at: https://www.ncbi.nlm.nih.gov/

● R (3.6) +scripts:
R package flowCybar: http://bioconductor.org/packages/release/bioc/html/flowCyBar.html

R package flowCHIC: http://www.bioconductor.org/packages/release/bioc/html/flowCHIC.html
R package ggplot2 and its smoothing function geom_smooth(…., method = “gam”, formula =
y ~ s(x, bs = “cs”)33

R script for the evaluation of the microscopic pictures (https://github.com/Allerdnec/InsightPro.git,

developed by C. Lepleux)
Reagent setup
PFA conservation solution
Dissolve 4 g of PFA in 50 ml of phosphate-buffered saline (PBS) (wt/vol). Heat the mix at 70 °C. Add
37% HCl solution (HCl, bidistilled water (vol/vol)) until the pH drops to 7.0. The PFA conservation
solution can be stored at 4 °C for 14 d or at −20 °C for a maximum of 6 months. Dilute this stock
PFA conservation solution to 2% with PBS for conservation of cells.
Ethanol fixation solution

70% pure ethanol in bidistilled water (vol/vol). The ethanol fixation solution can be stored at 4 °C for
14 d or at −20 °C for a maximum of 6 months.
PBS
Mix 6 mM Na2HPO4, 1.8 mM NaH2PO4 and 145 mM NaCl in bidistilled water, adjust to pH 7.0 and
autoclave. The buffer can be stored at 20 °C for a maximum of 6 months.
Permeabilization buffer
Mix 0.1 M citric acid and 4.1 mM Tween-20 in bidistilled water. The buffer can be stored at 4 °C for
4 weeks.
DAPI solution
Mix 0.24 µM DAPI and Na2HPO4/NaH2PO4 buffer (289 mM Na2HPO4 and 128 mM NaH2PO4
with bidistilled water, pH 7). The solution can be stored in the dark at 20 °C for at least
6 months.
SYBR Green I solution

Dilute a 10,000× stock with DMSO to a 200× solution and store it at −20 °C. For daily use,
dilute the 200× SYBR Green I with bidistilled water to a 4× working solution. Keep in darkness.
Add this solution to a sample to a final concentration of 0.1×. The solution must be freshly
prepared.
Ethidium bromide
Dilute 0.5 µg of ethidium bromide in 1 ml of bidistilled water. The solution must be freshly
prepared.
Difco gel solution

Dilute 1.8 g in 100 ml of bidistilled water (wt/vol) and autoclave for 20 min at 121 °C with bidistilled
water. The solution must be freshly prepared.
Sheath fluid (for MoFlo Legacy Cell Sorter)

Mix 10× stock solution of 19 mM KH2PO4, 38 mM KCl, 166 mM Na2HPO4 and 1.39 M NaCl with
bidistilled water to a 0.2× working solution. Autoclave stock solution for 20 min at 121 °C with
bidistilled water. The stock solution can be stored at 20 °C for a maximum of 6 months.
LB medium
Mix yeast extract 5 g l−1, NaCl 5 g l−1 and tryptone 10 g l−1 with bidistilled water and adjust to
pH 7.0. The solution must be freshly prepared.
LB agar medium
Use LB medium and add agar 20 g l−1. The solution must be freshly prepared.
Agarose
Mix 1.5 g of agarose with 100 ml of 0.5× TAE (wt/vol) and boil the mixtures thoroughly in a
microwave. Let the solution cool until it is hand hot; pour the mixture into a gel mold and wait until it
solidifies. The gel can be stored up to 1 week at 4 °C when wrapped in plastic film.

Difco gel
Warm the sterile Difco gel in a microwave until it becomes liquid. Apply 1 ml on the microscopic
glass slide and wait 3 min for the gel to polymerize. The solution must be freshly prepared.
TAE (0.5×)
Mix 40 mM Tris and 1 mM EDTA in bidistilled water and adjust to pH 8.0. The solution can be
stored at 20 °C for a maximum of 4 weeks.
Chelex 100 solution

Prepare 10% (wt/vol) with molecular-biology-grade water. Autoclave and store the solution at 20 °C
for a maximum of 4 weeks.
Mock community for sequencing MBARC26

Mock Bacteria Archaea Community is composed of 23 bacterial and three archaeal strains. All
genomes are sequenced20. The stock can be stored at −80 °C for a maximum of 12 months.
Equipment setup
BD Influx v7 Cell Sorter
The cell sorter is equipped with a stream-in-air nozzle, a blue 488-nm Sapphire OPS laser (400 mW)
and a 355-nm Genesis OPS laser (100 mW). Use the 488-nm laser for the analysis of the FSC
(488/10), the SSC (trigger signal, 488/10) and the SYBR Green I fluorescence (530/40). Use the
355-nm laser to excite the DAPI fluorescence (460/50). Detect light with PMTs (Hamamatsu R3896
PMTs in C6270 sockets). Run FACSFlow buffer fluidics at 33 p.s.i. through a 70-µm nozzle and detect
the cells equivalent to an event rate of 2,500–3,000 events per second. Calibrate the instrument daily
with 1-µm blue and 2-µm yellow-green FluoSpheres beads in the linear range.
MoFlo Legacy Cell Sorter

The MoFlo Legacy Cell Sorter is equipped with a stream-in-air nozzle, a blue 488-nm Genesis OPS
laser (tunable, 400 mW) and a 355-nm Xcyte laser (150 mW). Use the 488-nm laser for the analysis
of the FSC (488/10), the SSC (trigger signal, 488/10) and the SYBR Green I fluorescence (530/40). Use
the 355-nm laser to excite the DAPI fluorescence (450/65). Detect light with PMTs (R928, R3896).
Run sheath fluid at 56 p.s.i. through a 70-µm nozzle and detect the cells equivalent to an event rate of
2,500–3,000 events per second. Calibrate the instrument daily with 1-µm blue and 2-µm yellow-green
FluoSpheres beads in the linear range.
CyFlowSpace analyzer
The custom-made CyFlowSpace flow cytometer is equipped with a flow cuvette, a 355-nm Genesis
laser (60 mW) and a 488-nm Sapphire laser (50 mW). Use the 355-nm laser for the analysis of the
FSC (IPB 355 B) and the SSC (trigger signal, IPB 355 B) and to excite the DAPI fluorescence (460/50).
Use the 488-nm laser for the analysis of the SYBR Green I fluorescence (IPB 536/40 G). Detect
the light with PMTs (H6779-32, H6779-01). Run bidistilled water as sheath fluid with a flow rate of
0.5 µl s−1 during measurement, which is equivalent to an event rate of 500–800 events per second.
Calibrate the instrument daily with 0.5-µm and 1-µm blue BB Fluoresbrite beads and 2-µm yellow-
green FluoSpheres beads in the logarithmic range.
BD FACSVerse analyzer
The BD FACSVerse flow cytometer is equipped with a stainless steel flow cell and a blue 488-nm laser
(20 mW). Use the 488-nm laser for the analysis of the FSC (488/10), the SSC (trigger signal, 488/15)
and the SYBR Green I fluorescence (527/32). Detect the FSC with a Si-photodiode. Detect the SSC
and fluorescence with high-performance customized PMT modules. Run FACSFlow buffer as sheath
fluid with a flow rate of ~60 µl min−1. Calibrate the instrument daily using BD FACSuite CS&T
research beads.
BD Accuri C6+ analyzer

The BD Accuri C6+ flow cytometer is equipped with a flow cuvette and a blue 488-nm laser
(20 mW). Use the 488-nm laser for the analysis of the FSC (488/10), the SSC (488/15) and the SYBR

Green I fluorescence (trigger signal, 533/30). Detect forward and SSC with a Si-photodiode.
Detect the fluorescence with a fixed-voltage customized PMT module. Run the fluidics with Milli-Q
water at a constant flow rate of 66 µl min−1. Calibrate the instrument daily using BD CS&T
RUO beads.
AxioScope A1 fluorescence microscope

● The AxioScope A1 fluorescence microscope is equipped with an Illuminator HXP 120 V and a plan-
apochromat 100×/1.40 Oil DIC M27 objective. An Axiocam MRm camera and Axiovision software,
version 9.1, were used to visualize and measure the bacteria.
Software for cytometric data analysis

Most devices have their own software packages to record the data. Transfer the measured fcs files into
the latest FlowJo software package and evaluate the data by setting gates and defining the gate
template.
Procedure
Cultivation of the mCMC ● Timing Step 1, 3–5 d
1 Prepare plate-grown (Option A) or liquid-grown cultures (Option B) to make plate mCMC and
liquid mCMC, respectively. E. coli is not used for plate mCMC because the gates of this strain
overlap with the gates of P. polymyxa when grown on plates for 72 h. Option C describes how to
check the purity of the strains should this be necessary.
Handle the strains following the DSMZ’s recommendations, except that each single strain is
reactivated in LB medium
CRITICAL STEP For long-term storage, prepare each single strain as a glycerol stock. Mix 50%/
c
50% (vol/vol) LB medium culture and glycerol in cryotubes and store at −80 °C. Reactivate single
strains by redoing Step 1A(i) and 1B(i) for two times, respectively, before proceeding further to
avoid glycerol leftovers.
(A) Plate mCMC
(i) Streak K. rhizophila, P. polymyxa and S. rhizophila on LB agar plates, respectively, and
incubate at 30 °C for 72 h until harvest. Repeat this step (Fig. 1).
(B) Liquid mCMC
(i) Streak K. rhizophila, P. polymyxa, S. rhizophila and E. coli on LB agar plates, respectively,
and incubate at 30 °C for 72 h.
Prepare 100-ml flasks each containing 20 ml of LB medium.
(ii) Take single colonies as inocula and grow the strains separately at 30 °C at 150 r.p.m. for
24 h as pre-cultures.
(iii) Measure cell densities after 24 h. Calculate the respective milliliter amounts required to
achieve an OD(ʎ700nm = 0.5 cm) = 0.05 for Step 1B(iv).
(iv) Inoculate the appropriate amount of pre-culture into 500-ml flasks, each containing 100 ml
of LB medium, and grow the four strains separately at 30 °C at 150 r.p.m. for 24 h as main
cultures until harvest.
(C) (Optional) Testing purity of strains
CRITICAL It is not necessary to test the purity of the strains every time you make a new
c
mCMC. It is something that you do if you are concerned that there might be a problem. For
example, if you notice that there are different types of colonies on plates; if the slope of the
growth curve looks different to normal; or if the cells do not fit in the flow cytometry gate
template for the relevant species. You can analyze all strains as respective pure culture by using
flow cytometry to detect possible impurities.
(i) Collect one or two colonies of K. rhizophila, P. polymyxa, S. rhizophila and E. coli from
agar plates, respectively, or 1 ml from liquid solutions, respectively (see Step 2). Place
colonies separately into 2 ml of PBS. Place samples in microcentrifuge tubes and centrifuge
at 20,000g and 6 °C for 25 min; discard the supernatant carefully and re-suspend the cells
in 2 ml of PBS.
(ii) Dilute cells to an OD(dʎ700nm = 0.5 cm) = 0.01 with PBS and then transfer 70 µl of this
solution into microcentrifuge tubes. Centrifuge at 20,000g and 6 °C for 25 min; discard the
supernatant carefully and re-suspend the cells in 70 µl of Chelex 100 solution. Vortex for
10 s and incubate at 95 °C for 45 min in the Thermomixer.

(iii) Centrifuge the samples (5 min at 7,000g and 4 °C) and transfer 50 µl of the supernatant,
which contains the extracted DNA, to a new pre-chilled microcentrifuge tube and keep
it on ice
CRITICAL STEP Ensure that you do not transfer any Chelex beads with the supernatant,
c
as they will inhibit the PCR.
PAUSE POINT Store samples at −20 °C for a maximum of 6 months.
j
(iv) Perform the PCR with 20 pmol Forward Pro341F and Reverse Pro805R primers34, 4 nmol
dNTP, 4 µl of 5× Phusion GC solution, 40 nmol MgCl2, 0.4 units of Phusion High-Fidelity
Polymerase and 2 µl of DNA template. Add molecular-grade water to adjust the final
reaction volume to 20 µl. Use the PCR conditions as shown in the following table for 35
cycles. Add an initial 3-min denaturation step and a final 10-min extension step and keep
the product at 4°C. Purify and quantify the DNA using Agencourt AMPure beads XP-Kit.
Direction Primer sequence (5′>3′) Denaturation Annealing Extension

temperature
Forward CCTACGGGNBGCASCAG 95 °C, 30 s 52 °C, 45 s 72 °C, 45 s

(Pro341F) GACTACNVGGGTATCTAATCC
Reverse
(Pro805R)
PAUSE POINT Store purified amplicon solutions at −20 °C; solutions can be stored for
j
at least 6 months.
(v) Send the solutions to the sequencing company of your choice.
(vi) Analyze your sequencing results using a database of your choice and confirm the purity of
the strains.
Cell sampling and fixation ● Timing Step 2, 2h

CRITICAL Carry out all sampling, fixation and staining steps (Steps 2–4) in glass tubes, if possible.
c
Cells often adhere strongly to plastic tubes.

2 Harvest the cells and create a storable stock for future use. Either fix the cells with PFA and ethanol
(Option A) or dry them (Option B) (Fig. 1).
(A) Fixing with PFA/ethanol
(i) Harvest cells from LB agar plates with an inoculation loop and re-suspend them in 2 ml
of PBS. Only in case of P. polymyxa add 2 ml of PBS on the surface of the agar plate
and dissolve the cells using a Drigalski spatula and a pipette. Sonicate this cell suspension at
35 kHz and at 80 W for 5 min at 20 °C. Harvest cells from liquid cell cultures, taking either
a few milliliters or the whole 100 ml of cell suspension.
(ii) Centrifuge cell solutions at 3,200g for 10 min at 4 °C and discard the supernatant.
(iii) Wash the cells in 2 ml of PBS, adjust cell solutions to an OD(dʎ700nm = 0.5 cm) of
0.5 (~109–5 × 109 cells) and take 2 ml and centrifuge at 3,200g for 10 min at 4 °C and
discard the supernatant.
(iv) Stabilize cells in 2 ml of PFA solution for 30 min at room temperature. Vortex the sample
shortly. Centrifuge cell solutions at 3,200g for 10 min at 4 °C and discard the supernatant.
Finally, re-suspend the cells in 2 ml of ethanol fixation solution. Vortex the sample
shortly. Store the sample for at least 1 h before the cells are analyzed.
(B) Drying
(i) Prepare a heated vacuum centrifuge at −97 kPa and 490g (Supplementary Fig. 2)35.
(ii) Transfer the OD-adjusted cell suspensions in PBS into plastic tubes.
(iii) Centrifuge at 4,000g for 10 min at 4 °C, aspirate the supernatant carefully and dry the cells
at 35 °C for 45 min at 490g.
PAUSE POINT Keep ethanol-fixed cells at −20 °C and dried cells at 4 °C in the dark for a
j
maximum of 6 months.
? TROUBLESHOOTING
Cell staining ● Timing Steps 3 and 4, 30 min–12 h

3 Take an aliquot of an ethanol-fixed sample of strains, wash it with PBS (3,200g, 10 min, 4 °C) and
adjust the cells in PBS to an OD(dʎ700nm = 0.5 cm) of 0.04.

Re-suspend dried cells in 500 µl of PBS and vortex. Incubate these cells at room temperature for
30 min and vortex. Transfer these cells into glass tubes containing 2 ml of PBS. Sonicate for 2 min
and adjust cells in PBS to an OD(dʎ700nm = 0.5 cm) of 0.04.
4 Depending on the flow cytometer available, stain cells with either DAPI or SYBR Green I. DAPI
provides the higher resolution7 of the mCMC but requires a UV laser for excitation. SYBR Green I
provides less resolution36 of the mCMC, but almost all flow cytometers are equipped with a 488-nm
laser required for its excitation.
(A) Staining the mCMC with DAPI
(i) Take 2 ml of the OD-adjusted cell suspension, centrifuge (3,200g, 10 min, 4 °C) and
re-suspend the pellet in 1 ml of permeabilization buffer for 20 min at room temperature.
(ii) Centrifuge (3,200g, 10 min, 4 °C) and re-suspend the pellet in 2 ml of DAPI staining
solution for staining overnight in the dark.
(B) Staining the mCMC with SYBR Green I
(i) Take 100 µl of the OD-adjusted cell suspension, add PBS to get a total volume of 780 µl and
pre-incubate samples at 37 °C for 5 min.
(ii) Add 20 µl of SYBR Green I 4× working solution to reach a final concentration of 0.1× and
proceed the incubation at 37 °C for another 20 min in the dark
CRITICAL STEP Staining the strains not separately but jointly is also an option that
c
might save time. If you already know the ratios of the strains that you want to use for an
mCMC, mix the single strains of the plate or liquid mCMCs in their respective ratios
jointly into one tube and proceed immediately with Step 4 (A) or (B), followed by Step 6
(Supplementary Fig. 3).
? TROUBLESHOOTING
Construction and analysis of defined strain proportions in mCMCs ● Timing Steps 5–9,
30 min
5 Constitute either the plate or the liquid mCMC to defined strain proportions depending on the
application. To do this, mix different proportions of the strain suspensions obtained in Step 4.
The standard plate mCMC composition is P. polymyxa, S. rhizophila and K. rhizophila at a ratio of
80:1:19 (in vol/vol %), respectively.
The standard liquid mCMC composition is P. polymyxa, S. rhizophila, K. rhizophila and E. coli at a
ratio of 70:2.5:20:7.5 (in vol/vol %), respectively
CRITICAL STEP The optical density of larger cells (e.g., P. polymyxa) is higher than that of smaller cells
c
(e.g., S. rhizophila). This means that, if two cell suspensions have the same optical density, the one
containing smaller cells will have more cells in it. If you do not want to use the standard mCMC
compositions, make sure that the proportions of the respective strains are chosen according to application.
6 Filter the resulting mixtures using a 50-µm CellTrics filter to prevent clogging of the nozzle.
7 Add reference beads (0.5-µm and 1-µm) to the mCMCs as internal abiotic standards at a concentration of
~1% of events. Choose either blue or yellow-green fluorescent depending on whether the cells were stained
with DAPI or SYBR Green I.
? TROUBLESHOOTING
8 Use flow cytometry to analyze samples in terms of FSC, SSC and fluorescence. Use laser line 488 nm for
FSC, SSC and SYBR Green I excitation and the UV line for DAPI excitation. Create logarithmically scaled
2D plots.
9 Create a cell gate that must contain all cells of an mCMC and two gates for the reference beads. Use these
three gates for the measurements of the mCMC. The position of the beads in the gates must remain the
same. Measure ~200,000 cells per mCMC within the cell gate. If you want to analyze a single and pure
strain, the measurement of ~50,000 cells is sufficient
CRITICAL STEP Different applications of the mCMC might require an assembly where proportions of
c
the strains are different. Proportions can change the overall visualization of the cells in a 2D plot. Make
sure that the cells of all strains are clearly visible in the gate template after selecting the combinations
(Fig. 5 and Supplementary Tables 2–6).
(Optional) Cell counting ● Timing Step 10, 30 min

CRITICAL STEP Cell counting is an optional application. You might want to know precise cell
c
numbers per millileter instead of using proportions of strains adjusted to OD(dʎ700nm = 0.5 cm) of 0.04.

a 104
45:0.5:15:39.5 97.5:0.5:1.5:0.5 81.4:2.8:14.4:1.4 70:2.5:20:7.5

103
102
101
100
100 101 102 103 104
FSC (log)
b 104
80:19:1 75:1:24 53.3:4:42.7 80.1:19
103
102
101
100
100 101 102 103 104
FSC (log)
Fig. 5 | Construction of the liquid mCMC and plate mCMC at varying proportions. a,b Flow cytometric measurements of the mCMCs constructed out
of varying proportions of their species members corresponding to OD(dʎ700nm = 0.5 cm). The proportions are given (in %) within the 2D plots for P.
polymyxa, S. rhizophila, K. rhizophila and E. coli, respectively (a), and for P. polymyxa, S. rhizophila and K. rhizophila (b). Blue reference beads (0.5-µm and
1.0-µm) were spiked into the samples.
10 Perform cell counting with either DAPI- or SYBR Green I-stained cells using the cell
gate, respectively, of both the liquid and plate mCMCs (Figs. 2d and 3c and Supplementary
Methods 1)5,36.
Cytometric data analysis ● Timing Steps 10–15, 30 min

11 Load the cytometric data into the FlowJo software package and create a gate template. Exclude
beads and instrumental noise. Mark all subcommunities with an ellipsoid gate. Create non-
overlapping gates for the most abundant subpopulations. Here, the gate templates were created for
plate mCMC (Option A) and liquid mCMC (Option B) based on measurements of the mCMC with
the BD Influx v7 Cell Sorter and UV excitation.
(A) Plate mCMC
(i) Set gates for the most abundant subpopulations: S. rhizophila (P1 to P3), K. rhizophila
(P4 to P6) and P. polymyxa (P7 to P11) (Fig. 3c and Supplementary Table 2).
(ii) Form the gate template using gates P1 to P11 for the plate mCMC.
(B) Liquid mCMC
(i) Set gates for the most abundant subpopulations: S. rhizophila (L1 and L2), E. coli
(L3 and L4), K. rhizophila (L5 to L7) and P. polymyxa (L8 to L12) (Fig. 2d and Supple-
mentary Table 4).
(ii) Form the gate template using gates L1 to L12 for the liquid mCMC.
? TROUBLESHOOTING
12 Form complementary gate templates for the SYBR Green I-stained cells for liquid and plate
mCMCs (Supplementary Fig. 4).
13 As a standard procedure, routinely fit the selected mCMC (either plate or liquid and in terms
of strains proportions) into its gate template before subsequently measuring microbial
communities
CRITICAL STEP Use the mCMCs also for measurement campaigns in which flow cytometers
c
of other brands are operated. Depending on the resolution qualities of a cytometer, the number

Box 1 | Microscopic analysis of sorted cells
Procedure ● Timing 4h–1 d

1 Prepare glass slides by coating them freshly and thinly with Difco gel and adding 1–3 µl of cells. Wait 3 min for
the cells to be fixed on the gel surface and the liquid to evaporate before carefully placing the coverslip on top.
This will prevent active and passive movement of cells
CRITICAL STEP Sometimes air bubbles might remain under the coverslip. Press lightly on the coverslip to
c
replace the air with the DAPI staining solution.
2 Switch on microscope and connected camera as well as computer and microscope software.
3 Place the glass slide on the microscope stage and adjust the focus. Preferably, use an immersion objective
(×100 magnification) and immersion oil
CRITICAL STEP Make sure that a single-cell layer is prepared for microscopic analysis.
c
4 Analyze the width and length of at least 500 different cells using a dedicated program (here: Insight Pro 9.1).
CRITICAL STEP This analysis step can take several hours. Make sure that the sample can be analyzed
c
within 1 d.
5 List all cell data in a table. If Insight Pro 9.1. was used, evaluate size distributions with an R script (https://
github.com/Allerdnec/InsightPro.git).
of separately identifiable subpopulations can vary. In addition, the position of the subpopu-
lations in a 2D plot can be different. Examples for DAPI- and SYBR Green I-stained, analyzed
and gated cell subsets of the liquid and plate mCMCs are presented in Fig. 4b and Supplementary
Fig. 1.
? TROUBLESHOOTING
14 Use the mCMC as a quality marker before comparing measurement series from different
measurement days and locations. If the mCMC always matches the gate template, the measured
data from microbial communities can be evaluated by, for example, FlowJo statistics.
? TROUBLESHOOTING
15 Use, for example, the flowCHIC algorithm for dissimilarity analysis to compare the plate
mCMC and the liquid mCMC and the respective triplicate measurements (see, for example,
Fig. 2c). Similarly, the flowCyBar algorithm can be used on the basis of the gate template.
Use one of the two tools to confirm similarity of the mCMC distributions over measure-
ment days.
Cell sorting of mCMC gates ● Timing Step 16, 30 min–3 h

16 Prepare the flow cytometer for stable sorting using the appropriate settings explained in the
manufacturer’s manual (e.g., by determining the correct drop delay). Maintain these settings for all
samples of an experiment. Select a subpopulation from the mCMC, which might be a single gate, an
association of gates or the entire cell gate, and sort, using a cell sorter, at least 500,000 cells into
microcentrifuge tubes. In this protocol, the sorting was done by the BD Influx v7 Cell Sorter using
the four-way-sort option and the ‘1.0 Drop Pure’ sort mode at event rates between 3,000 and 10,000
events per second
CRITICAL STEP If numbers of cells per gate are low, proceed the sorting on the next day.
c
? TROUBLESHOOTING
17 Sort quality. To check the quality of the cell-sorting method by flow cytometry, sort subpopulations
of cells, centrifuge at 3,200g at 4 °C for 10 min and re-suspend in 100 µl of DAPI staining solution
and immediately re-measure the sorted cell fraction. The cells should appear within the identical
gate template from that they were taken from (Fig. 3a).
18 Microscopy. To find out the morphology (cell stage) of the sorted cells, sort at least 1,000,000 cells
per gate, centrifuge at 3,200g at 4 °C for 10 min and re-suspend in 2–5 µl of DAPI staining
solution. In case of liquid-grown P. polymyxa cells, sort the cells directly on microscopic
slides without centrifugation. For microscopic visualization and evaluation, proceed as described
in Box 1.
19 Illumina amplicon sequencing. To identify the cell population or cell subpopulation by DNA
sequencing, sort 500,000 cells per gate. Centrifuge the cells at 20,000g at 6 °C for 25 min.
PAUSE POINT Store pellets at −20 °C for a maximum of 6 months.
j
20 For preparing the cells for Illumina amplicon sequencing, proceed as described in Box 2.
21 For evaluating the sequencing data, proceed as described in Supplementary Methods 1.

Box 2 | Illumina amplicon sequencing of sorted cells
Procedure ● Timing 1–3 d
1 Add 70 µl of Chelex 100 solution to each frozen cell pellet.
2 Add 300 µl of Chelex 100 solution to every control sample pellet.
CRITICAL STEP Include positive controls: 1 ml of fluorescently stained and unsorted cells as well as 1 ml of unstained and unsorted cells to
c
determine community structure of the whole community and to test the influence of the staining procedure on community structure. Include
the MBARC26 as sequencing mock community. Process the positive controls as the other samples.
CRITICAL STEP Include negative controls: 1 ml of empty sheath buffer and an empty tube. Process the negative controls as the other samples.
c
3 Vortex for 10 s, incubate at 95 °C for 45 min and centrifuge at 7,000g and 4 °C for 5 min.
4 Transfer 50 µl of the supernatant, which contains the extracted DNA, to a new pre-chilled tube and keep it on ice.
CRITICAL STEP Ensure that you do not transfer any Chelex beads with the supernatant, as they will inhibit the PCR.
j c
PAUSE POINT Samples can be stored at −20 °C for several months.

5 Test DNA quality by Qubit 3.0.
CRITICAL STEP Extracted DNA from sorted cells might be too low in quantity to be detected by Qubit 3.0. Perform a PCR with 35 cycles
c
using the primers Forward 27F and Reverse 1492R following the recommendations of Lane et al.34.
6 Set up a library as listed below and follow the manufacturer’s instructions.
Reaction component Amount per reaction Final concentration
Negative control (µl) (Un-)sorted cells (µl)
Nuclease-free water 0 9.8

5× Phusion GC solution 4 4 1×
MgCl2 1.6 1.6 2 nmol µl−1
dNTP mix 0.4 0.4 0.2 nmol µl−1
Primer 1 Pro341F (10 pmol µl−1) 1 1 0.5 pmol µl−1
Primer 2 Pro805R (10 pmol µl−1) 1 1 0.5 pmol µl−1
Polymerase Phusion HF 0.2 0.2 0.02 units µl−1
DNA template 11.8 2 0.1 units µl−1
7 Use a dual-indexing approach to allow the pooling of multiple 16S rRNA gene amplicon libraries of the V3-V4 region in a single MiSeq run. To
do this, perform a two-step PCR.
8 PCR 1. Use the non-indexed forward and reverse primers V3-V4 Pro341F37 and Pro805R38 for 20 cycles (for sorted cells, 22 cycles) within the
following conditions (cycle Steps 1–23).
? TROUBLESHOOTING
Mother primer 1–2
Step Denature Anneal Extend Hold
1 3 min at 95 °C
2–21 30 s at 95 °C 45 s at 52 °C 45 s at 72 °C
22 10 min at 72 °C
23 4 °C
9 Test success of PCR in the control samples using standard agarose gel electrophoresis (1.5% agarose with 0.5× TAE (wt/vol)). Mix 3 µl of PCR
product with 2 µl of 0.5× TAE and 1 µl of 6× loading dye. Load and run the agarose gel for 30 min at 100 V. Stain the agarose gel with ethidium
bromide staining solution for 5 min and analyze with a gel documentation system.
10 If the controls are correct, purify the PCR product (proceed at Step 23). Mix 10 µl of PCR product with 18 µl of AMPure XP for 3 min at room
temperature to bind DNA fragments to paramagnetic beads. Place the reaction tube onto a magnetic separation rack for 5 min and discard the
clear solution containing the PCR reagents without any amplicons. Wash the beads and their bound DNA fragments with 70% ethanol to
remove potential contaminants. Resuspend the beads in 10 µl of nuclease-free water for 1 min at room temperature. Collect the purified DNA
fragments from the clear solution after placing the tube on the magnetic separation rack for 1 min.
11 PCR 2. Use six-nucleotides-barcoded mother primers for 8–10 cycles (Steps 24–35), dependent on the purified DNA template quality and
quantity (proceed from Step 9). Process negative controls for 35 cycles to ensure quality and purity of the prepared barcoded libraries.
Barcoded mother primer 1–2
Step Denature Anneal Extend Hold
24 3 min at 95 °C
25–33 30 s at 95 °C 45 s at 52 °C 45 s at 72 °C
34 10 min at 72 °C
35 4 °C

Box 2 | Illumina amplicon sequencing of sorted cells (continued)

12 Purify the PCR products from Step 35 as described above (Step 9 of Box 2). Quantify the DNA in the PCR fragment solution using Qubit 3.0 and
the dsDNA HS Assay Kit.
PAUSE POINT Store PCR products by freezing at −20 °C. Samples can be stored for several months before being sent to a sequencing
j
company.
13 Pool equimolarly and send to your sequencing company of choice.
? TROUBLESHOOTING
14 Analyze your sequencing data set with your preferred bioinformatics pipeline.
In this protocol, the sequencing data sets were analyzed by mothur 1.41 (ref. 31) and R 3.6 using the Silva database v132 and compared to the
Sanger sequencing data (Supplementary Methods 1).
Troubleshooting
Troubleshooting advice can be found in Table 1.
Table 1 | Troubleshooting table
Step Problem Possible reason Solution
2 Other fixation method available Good experience with other We recommend fixing the strains of the mCMC using the
fixation method other fixation method
Make sure that the other method reliably fixes the cells
4 Other staining procedures available Use of higher or lower amounts of We recommend staining the strains of the mCMC using
SYBR Green I or DAPI or use of the other staining procedure
other staining procedures Make sure that the other procedure resolves the
cytometric patterns of the mCMC in at least three or four
subpopulations
Resolution and position of cells in Different experimenters handle the We recommend to use the mCMC daily. Make sure that
the 2D plot is of different quality cells and the cytometers with the mCMC fits into the gate template. If not, repeat Steps
within the same experiment different skills 2–9. The mCMC can also be used to train experimenters
on cytometers
7 The mCMC cannot be differentiated Bad alignment of the cytometer or Align the cytometer with 0.5-µm or 1-µm beads first.
from noise cells are not stained Make sure that the trigger has been set on the SSC.
Make sure that the instrument is not blocked and the
tubings are clean. Test the correct staining of cells by
microscopy
11 No FlowJo software package No money to buy one Use on-machine software or open-source software such
available as FCSalyzer (https://sourceforge.net/projects/fcsa
lyzer/) or CytoSpec. (http://www.cyto.purdue.edu/
cytospec)
13 Cells of the mCMC do not fit into the Contamination of strains, no use of Test strains for purity
gate template stationary phase cells, incorrect Make sure that cells are harvested in the stationary
fixation and staining of cells, phase of growth
incorrect assembly of mCMC or Make sure that strains are fixed and stained according to
too low or too high laser power the protocol
Make sure that all strains are part of the mCMC. Test
stability of laser power
Exemplary tests are listed in Supplementary Tables 2–6
Proportions of cells in the gate Contamination of strains Test strains for purity
template change
Cells of the mCMC cannot be Low resolution of the cytometer or Ensure instrumental settings by beads, ensure correct
differentiated by the gate template incorrect fixation and staining fixation and staining by measuring the single strains of
the mCMC and test resolution of the device by
assembling low versus high cell numbers per strain
14 Users might want to compare data Cooperation between labs, logistic A direct comparison of data is only possible if flow
between labs reasons cytometers of the same brand are used. We recommend
using the mCMC to test the comparability of flow
cytometers
16 No DNA of sorted cells Loss of cells during centrifugation Use unsorted mCMC as control sample. Dilute unsorted
after sorting cells to an OD(dʎ700nm = 0.5 cm) of 0.01 in 70 µl of PBS
before the DNA extraction. Pellet the cells for 25 min at
20,000g and 6 °C
Table continued

Table 1 (continued)
Step Problem Possible reason Solution
Box 2 Wrong PCR products Contaminations during PCR Run negative and positive PCR controls for 35 cycles
(Step 8) (Box 2). Replace PCR reagents and the DNA template by
water in the negative controls. Check these controls with
a standard agarose gel electrophoresis to ensure that the
PCR was free of contamination
Box 2 Unsuccessful sequencing Technical biases Perform every PCR in triplicate and purify and quantify
(Step 13) separately before pooling the triplicates as a unique
sample after both PCR steps and DNA quantification.
Process the MBARC26 in the same way to ensure the
quality of the PCR steps and the following evaluation
Timing
It takes most of the time to cultivate the four strains. Once the strains are harvested and fixed, they
are ready for everyday use. A stock of each strain should be produced that can be used for 6 months.
After staining, all of the following steps can be carried out in a few minutes. Sorting cells takes longer,
depending on the proportion of cells in a sorted gate.
Step 1, cultivation of the mCMC: 3–5 d, depending on plate or liquid mCMC
Step 2, cell sampling and fixation: 2 h
Steps 3 and 4, cell staining: 30 min–12 h, depending on using SYBR Green I or DAPI
Steps 5–9, construction and analysis of defined strain proportions in mCMCs: 30 min
Step 10, cell counting: 30 min
Steps 11–15, cytometric data analysis: 30 min
Step 16, cell sorting of mCMC gates: 30 min–3 h, depending on how abundant cells are in a gate
Box 1, microscopic analysis of sorted cells: 4 h–1 d, depending on how many cells can be sorted on a slide
Box 2, Illumina amplicon sequencing of sorted cells: 1–3 d
Anticipated results
Microbial communities are highly variable in their structure. This is a result of short generation times
of their members, interactions of cells in communities, stochastic events or abiotic factors5. Microbial
community flow cytometry visualizes changes in the assembly of communities through dense
sampling campaigns and measurements. The evaluation of the data can then be done almost on-line
by using established bioinformatics tools7. The standardization of data generation is a crucial step in
this endeavor, because even small deviations in the patterns and positions of cells in a 2D plot of two
consecutive samples can be detected and will influence the interpretation of data.
Frequently, a flow cytometer must be aligned by beads of the size of bacteria positioned always in
the same gate in a 2D plot. An example for not using the same gates is shown in Supplementary
Methods 2. Such different settings of a flow cytometer make it impossible to compare samples from
microbial communities using bioinformatics tools.
The mCMC ensures accurate sample handling. An altering treatment of microbial communities
changes the position of cells in a 2D plot. To demonstrate the effect of altering cell treatments, the plate
mCMC was used as standard. In addition, three communities from activated sludge, digester fluid and
digester centrate of a sewage plant were used. The cells of the three communities were handled as described
in Supplementary Methods 2 (SYBR Green I staining) and Supplementary Methods 3 (DAPI staining).
For the plate mCMC, the gate template of Fig. 3c was used, which comprised 11 gates (P1 to P11)
containing the mock strains and their subpopulations S. rhizophila (P1 to P3), K. rhizophila (P4 to
P6) and P. polymyxa (P7 to P11). The spores of P. polymyxa were marked by gates P10 and P11. The
additional off-gate comprises all cells outside the gate template but inside the cell gate. A community
fingerprint highlighted increasing cell numbers in gates in dark blue and decreasing in light blue
(Fig. 6). All samples represent independently handled parallels of the plate mCMC treated according
to the routine procedure but with one step changed each time (Fig. 6, Supplementary Methods 3).
First, the influence of varying cell numbers in samples on comparability in fluorescence intensity of
cells between samples was tested by analyzing cell solutions of the plate mCMC with OD(dʎ700nm = 0.5 cm)
values between 0.01 and 0.4. An increased number of cells bound less dye per cell and shifted the cells

mCMC Activated sludge Digester fluid Centrate
OD = 0.01
Changes in
cell density OD = 0.04
OD = 0.4
−PB
DAPI concentration
Changes in
0.24 µM DAPI
Normalized
relative 0.68 µM DAPI
abundance
2
1 µM DAPI
1.6
1.2
0.8
centrifugation speed
500 g
Changes in
0.4
0
3,200 g
20,000 g
Overnight staining
staining time
Changes in
1 h staining
10 min staining
1 min staining
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
Off−gate
G1
G2
G3
G4
G5
G6
G7
G8
G9
Off−gate
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
G13
G14
G15
G16
G17
Off−gate
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
G13
Off−gate
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. By using the tool flowCyBar, possible effects are shown that change numbers of cells
in the gates of the gate template, such as changes in cell density (OD = 0.01, OD = 0.04 and OD = 0.4), DAPI concentration (cell staining without
pre-treatment of permeabilization buffer (−PB), 0.24 µM DAPI, 0.68 µM DAPI and 1 µM DAPI), centrifugation speed (500g, 3,200g and 20,000g)
and staining time (overnight, 1 h, 10 min and 1 min). The standard cell treatstment conditions are highlighted in black.
from one gate (e.g., P3 and P6) to other gates (e.g., P2 and P5, P9 from off-gate cells), which was also
reflected in the altered fingerprint of the plate mCMC (Fig. 6). Second, increasing concentrations from
standard 0.24 µM to 1 µM DAPI shifted the position of the cells even further in the 2D plot, which
resulted in nearly empty gates (P2, P4, P5, P7 and P11) and a huge increase in the cell number of off-gate
cells (Fig. 6 and Supplementary Methods 3). Third, we also tested whether centrifugation speed could
affect the community fingerprint by using speeds of 500–20,000g. Here, the effect was not as strong, but we
lost cell numbers in the small gates (P1 to P3) at low centrifugation rates. At elevated speed, cell numbers
in some gates increased considerably, whereas others decreased (e.g., K. rhizophila, P4 to P6). Finally, we
compared different staining times starting from 1 min to 10 min and 1 h to the 12 h (overnight) of the
standard procedure. The short staining time surprisingly covered the strains S. rhizophila (P1 to P3) and K.
rhizophila (P4 to P6) but the cells, and in particular the spores, of P. polymyxa (P10 and P11) moved from
both their gates and even out of the cell gate (Fig. 6 and Supplementary Methods 3). Nevertheless, the
resolution remained high even for only 1-min staining time, which could qualify the method for on-line
applications. In this case, we recommend the setting up of a new cell gate and a new gate template. It
should be noted that the bead combination of 0.5-µm and 1-µm beads did not change their position in all
applications, which points for a stable setting of the cytometer but also shows that variations in sample
treatment cannot be detected without a community standard, such as the mCMC.
The same tests were carried out for the three other communities (Supplementary Methods 3). The
activated sludge, the digester fluid and the digester centrate communities were treated according to the
standard procedure for the plate mCMC (Fig. 6). The communities from activated sludge distributed
into nine gates, from digester fluid into 17 gates and from centrate into 13 gates. The patterns of the
three natural communities in the 2D plots were most affected by changes in cell numbers in the cell
solutions. An increased OD(dʎ700nm = 0.5 cm) caused cells to shift out of the gate template and to increase
the number of off-gate cells within the cell gate. The second most important variation was the increase
in the DAPI concentration to 1 µM, which shifted cells from the original gates to higher positions of the
2D plots and thus foreign gates (Fig. 6 and Supplementary Methods 3), which originally belonged to
other subcommunities. The low centrifugation speed caused only a few changes, similar to the
experience obtained by testing this variation with the plate mCMC. The influence of the staining time

on cell distributions was also not as severe as the first two variations tested (Supplementary Methods 3).
The influence of various cell handling on cell pattern was also investigated for SYBR Green I-treated
microbial communities (Supplementary Methods 2), which showed similar results.
Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary
linked to this article.
Data availability
The cytometric data sets generated during the current study are available under FlowRepository:
https://flowrepository.org/, no. FR-FCM-Z2CJ. The Illumina MiSeq data sets generated during the
current study are available under BioProject: https://www.ncbi.nlm.nih.gov/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: http://bioconductor.org/packages/release/bioc/html/flowCyBar.html.
R package flowCHIC: http://www.bioconductor.org/packages/release/bioc/html/flowCHIC.html.
R script for the evaluation of the microscopic pictures (https://github.com/Allerdnec/InsightPro.git,
developed by C. Lepleux).
References
1. Müller, S. & Nebe-von-Caron, G. Functional single-cell analyses: flow cytometry and cell sorting of microbial
populations and communities. FEMS Microbiol. Rev. 34, 554–587 (2010).
2. Günther, S. et al. Species-sorting and mass-transfer paradigms control managed natural metacommunities.
Environ. Microbiol. 18, 4862–4877 (2016).
3. Props, R., Monsieurs, P., Mysara, M., Clement, L. & Boon, N. Measuring the biodiversity of microbial
communities by flow cytometry. Methods Ecol. Evol. 7, 1376–1385 (2016).
4. Liu, Z. et al. Ecological stability properties of microbial communities assessed by flow cytometry. mSphere 3,
e00564–17 (2018).
5. Liu, Z. et al. Neutral mechanisms and niche differentiation in steady-state insular microbial communities
revealed by single cell analysis. Environ. Microbiol. 21, 164–181 (2019).
6. De Vrieze, J., Boon, N. & Verstrate, W. Taking the technical microbiome into the next decade. Environ.
Microbiol. 20, 1991–2000 (2018).
7. Koch, C. et al. Cytometric fingerprinting for analyzing microbial intracommunity structure variation and
identifying subcommunity function. Nat. Protoc. 8, 190–202 (2013).
8. Mage, L. M. et al. Shape-based separation of synthetic microparticles. Nat. Mater. 18, 82–89 (2019).
9. Müller, S. Modes of cytometric bacterial DNA pattern: a tool for pursuing growth. Cell Prolif. 40, 621–639
(2007).
10. Ludwig, J., Höner zu Siederdissen, C., Liu, Z., Stadler, P. F. & Müller, S. flowEMMi: an automated model-
based clustering tool for microbial cytometric data. BMC Bioinforma. 20, 643 (2019).
11. Koch, C., Fetzer, I., Harms, H. & Müller, S. CHIC-an automated approach for the detection of dynamic
variations in complex microbial communities. Cytom. A 83, 561–567 (2013).
12. Liu, Z. & Müller, S. Bacterial community diversity dynamics highlight degrees of nestedness and turnover
patterns. Cytom. Part A https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.23965 (2020)
13. Aghaeepour, N. et al. Critical assessment of automated flow cytometry data analysis techniques. Nat. Methods
10, 228–238 (2013).
14. Peters, J. M. & Ansari, M. Q. Multiparameter flow cytometry in the diagnosis and management of acute
leukemia. Arch. Pathol. Lab. Med. 135, 44–54 (2011).
15. Bendall, S. C. et al. Single-cell mass cytometry of differential immune and drug responses across a human
hematopoietic continuum. Science 332, 687–696 (2011).
16. Spitzer, H. M. & Nolan, G. P. Mass cytometry: single cells, many features. Cell 165, 780–791 (2016).
17. Overmann, J., Abt, B. & Sikorski, J. Present and future of culturing bacteria. Annu. Rev. Microbiol. 8, 711–730
(2017).
18. Nayfach, S., Shi, Z. J., Seshadri, R., Pollard, K. S. & Kyrpides, N. C. New insights from uncultivated genomes
of the global human gut microbiome. Nature 568, 505–510 (2019).
19. Roesch, L. F. et al. Pyrosequencing enumerates and contrasts soil microbial diversity. ISME J. 1, 283–290
(2007).
20. Singer, E. et al. Next generation sequencing data of a defined microbial mock community. Sci. Data 3, 160081
(2016).
21. Hallmaier-Wacker, L. K., Lueert, S., Roos, C. & Knauf, S. The impact of storage buffer, DNA extraction
method, and polymerase on microbial analysis. Sci. Rep. 8, 6292 (2018).

22. Hardwick, S. A. et al. Synthetic microbe communities provide internal reference standards for metagenome
sequencing and analysis. Nat. Commun. 9, 3096 (2018).
23. Hornung, B. V. H., Zwittink, R. D. & Kuijper, E. J. Issues and current standards of controls in microbiome
research. FEMS Microbiol. Ecol. 95, fiz045 (2019).
24. Sze, M. A. & Schloss, P. D. The impact of DNA polymerase and number of rounds of amplification in PCR
on 16S rRNA gene sequence data. mSphere 4, e00163–19 (2019).
25. Clingenpeel, S., Clum, A., Schwientel, P., Rinke, C. & Woyke, T. Reconstructing each cell’s genome within
complex communities—dream or reality? Front. Microbiol. 8, 771 (2015).
26. Stepanauskas, R. et al. Improved genome recovery and intergrated cell-size analyses of individual uncultured
microbial cells and viral particles. Nat. Commun. 8, 84 (2017).
27. De Bruin, O. M. & Birnboim, H. C. A method for assessing efficiency of bacterial cell disruption and DNA
release. BMC Microbiol. 16, 197 (2016).
28. Mie, G. Beiträge zur optik trüber medien, speziell kolloidaler metallösungen. Ann. Phys. 25, 377–445 (1908).
29. Woyke, T., Doud, D. F. R. & Schulz, F. The trajectory of microbial single-cell sequencing. Nat. Methods 14,
1045–1054 (2017).
30. Jahn, M. et al. Subpopulation-proteomics in prokaryotic populations. Curr. Opin. Biotech. 24, 79–87 (2013).
31. Schloss, P. D. et al. Introducing mothur: open-source, platform-independent, community-supported software
for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009).
32. Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C. & Knight, R. UCHIME improves sensitivity and speed of
chimera detection. Bioinformatics 27, 2194–2200 (2011).
33. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, 2016).
34. Lane, D. J. in Nucleic Acid Techniques in Bacterial Systematics (eds. Stackebrandt, E. & Goodfellow, M.)
115–175 (Wiley, 1991).
35. Lambrecht, J. et al. Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on
F420 autofluorescence. Microb. Cell Fact. 16, 180 (2017).
36. Besmer, M. D. et al. The feasibility of automated online flow cytometry for in-situ monitoring of microbial
dynamics in aquatic ecosystems. Front. Microbiol 5, 265 (2014).
37. Takahashi, S., Tomita, J., Nishioka, K., Hisada, T. & Nishijima, M. Development of a prokaryotic universal primer
for simultaneous analysis of bacteria and archaea using next-generation sequencing. PLoS ONE 9, e105592 (2014).
38. Herlemann, D. P. et al. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic
Sea. ISME J. 5, 1571–1579 (2011).
Acknowledgements
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 (https://github.com/Allerdnec/InsightPro.git) and Z. Liu for creating Fig. 6 and Supplementary Methods 3.
Author contributions
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.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41596-020-0362-0.
Correspondence and requests for materials should be addressed to S.M.
Reprints and permissions information is available at www.nature.com/reprints.
Received: 9 January 2020; Accepted: 27 May 2020;

Published online: 7 August 2020
Related links
Key references using this protocol
Ludwig, J., Höner zu Siederdissen, C., Liu Z, Stadler, P.F. & Müller, S. BMC Bioinformatics 20, 643 (2019):
https://doi.org/10.1186/s12859-019-3152-3
Liu, Z. & Müller, S. Cytom. Part A (2020): https://doi.org/10.1002/cyto.a.23965

nature research | reporting summary
Corresponding author(s): Susann Müller
Last updated by author(s): Mar 2, 2020
Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency
in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.
Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement
A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly
The statistical test(s) used AND whether they are one- or two-sided
Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested

A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons
A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient)
AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)
For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted
Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes
Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated
Our web collection on statistics for biologists contains articles on many of the points above.
Software and code

Policy information about availability of computer code
Data collection NCBI data base (Bethesda MD, National Library of Medicine US, National Center for Biotechnology Information, 1988) – (cited 2019 Nov
25). Available from: https://www.ncbi.nlm.nih.gov/
FlowRepository: https://flowrepository.org/, number: FR-FCM-Z2CJ
Data analysis R (3.6) +scripts:

R package flowCybar: http://bioconductor.org/packages/release/bioc/html/flowCyBar.html
R package flowCHIC: http://www.bioconductor.org/packages/release/bioc/html/flowCHIC.html
R package ggplot2 and its smoothing function geom_smooth(…., method = "gam", formula = y ~ s(x, bs = "cs")31
R script for the evaluation of the microscopic pictures (https://github.com/Allerdnec/InsightPro.git)
UCHIME
Mothur 1.4129
FlowJoTM 10.0.8.r1 (FlowJo, Tree Star / BD)
Image-Insight Pro 9.1 (Media Cybernetics, Inc.)
Silva SSU / LSU database v132 (https://www.arb-silva.de/)
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers.
October 2018
We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.
1
Data

Policy information about availability of data
All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:
- Accession codes, unique identifiers, or web links for publicly available datasets
- A list of figures that have associated raw data
- A description of any restrictions on data availability
The cytometric datasets generated during the current study are available under FlowRepository: https://flowrepository.org/, number: FR-FCM-Z2CJ. The
IlluminaMiSeq datasets generated during the current study are available under BioProject: https://www.ncbi.nlm.nih.gov/bioproject, accession number:
PRJNA541369.
Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf
Ecological, evolutionary & environmental sciences study design

All studies must disclose on these points even when the disclosure is negative.
Study description We developed an artificial microbial Cytometric Mock Community (mCMC) as a reference standard and benchmark to support the
generation of dynamic, high quality cytometric microbial community data. Our approach upholds quantitative assessments and
qualified decisions for sorting of subcommunities suitable for subsequent high resolution sequencing or proteomic routines.
This protocol provides an artificial microbial community of known structure that can be effectively used as a daily standard for
routine flow cytometric measurements of natural microbial community samples from different environments. This standard is
recommended to be composed of either 3 or 4 pure strains at different proportions which can be well separated by gates in a 2D-
plot. The populations show further heterogeneity due to subpopulations, which are marked by additional gates. The subpopulations
arise from different microbial cell cycle states or life cycle states and should appear at multiple, fixed positions in a 2D-plot.
A mCMC is helpful in several ways:
1. With the mCMC, the position and the resolution range of bacteria can be adjusted in all commercially available flow cytometers
2. Together with beads in the range of the microbial cells the flow cytometers can always be precisely aligned into the identical
standard position
3. The operator-dependent fluctuations in cell treatment (sampling, fixation, staining) are minimized
4. Measuring data over long periods of time is no longer a problem (especially important for measuring community dynamics)
5. The comparison of data between laboratories is now possible
6. Small deviations in the cytometric patterns, which normally occur without using an mCMC, do not distort data created with
automatic bioinformatics pipelines
7. The mCMC serves as test microbial community for the development of bioinformatics tools
8. The mCMC is an artificial community, the members of which can be composed in different proportions or in different compositions
depending on the application
9. The mCMC serves as test community for correct cell sorting/back-measurement
10. The mCMC serves in any subsequent method (e.g. after sorting) as marker for correct cytometric experiment set up.
Standardization is an important issue in microbial community flow cytometry. The open source programs for automatic evaluation of
cytometric microbial community data are very sensitive and mark the slightest differences between two subsequently measured
community samples.
Research sample For the mock community non-pathogenic bacteria were selected which are readily available and cultivable. Both Gram+ and Gram-
strains with known GC contents and fully sequenced genomes were included. Based on these criteria, Stenotrophomonas rhizophila
DSM 14405, Escherichia coli DSM 4230, Kocuria rhizophila DSM 348, and Paenibacillus polymyxa DSM 36 were selected
(Supplementary Table 1).
Strain cultivation: Two types of mCMCs were constructed. The liquid-grown mCMC is called liquid mCMC and contains all four strains.
The mCMC grown on plate alone is called plate mCMC and contains the strains S. rhizophila DSM 14405, K. rhizophila DSM 348, and
P. polymyxa DSM 36.
From a sewage plant three natural microbial communities were sampled from three different locations such as activated sludge,
digester fluid, and centrate and cultivated.
October 2018
Sampling strategy Three biological parallels, each, were taken for the determination of OD values of samples from the growth curves. The same
samples were used for flow cytometry. The pure strains were assembled together at varying known proportions, differentially
treated and cytometrically measured. The proportions of strains (and subpopulations) were re-evaluated counting cell numbers by
FlowJo. Cell sizes were re-evaluated by fluorescence microscopy. The presence of the strains in the mock community were evaluated
by Illumina-MiSeq sequencing. In addition, the purity of sorted subpopulations were evaluated by microscopy and Illumina-MiSeq
sequencing. The comparison of flow cytometric data was done by using biological parallels in addition to technical parallels by using
flowCHIC (using Bray-Curtis-based non-metric multidimensional scaling (NMDS) of 21 samples (200 iterations).
The same samples were measured on different flow cytometers.
2
Wastewater samples were precultured as described and measured in parallel at different flow cytometers and using different
treatments.

Data collection 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.
Timing and spatial scale The mCMC samples were measured between March 2018 and December 2019. Cell sorting was performed in July 2018 and both the
microscopy and the sequencing data were obtained afterwards. All dates of samples measured by flow cytometry are provided as
part of the 2D-plot information in the flow repository.
The data for the wastewater samples (for Anticipated results) origined from different locations such as activated sludge, digester
fluid, and centrate. These samples were measured between September to December 2019.
Data exclusions Only for the sequencing analysis data were excluded: Following the Bokulich guidelines we set the OTU threshold to 0.0447 % in
order to exclude sequences below this abundance threshold for further evaluation.
Reproducibility For the sequencing data triplicate samples were used and negative and positive controls were included (see Box2). The standard
MBARC26 was processed in the same way as the samples to ensure the quality of the PCR steps and the following evaluation
procedure. In addition, subpopulations were sorted and their composition verified by Illumina-MiSeq sequencing.
For microscopy at least 500 cells were measured.
For flow cytometry we always use beads of sizes between 0.5 μm and 2 μm:
Reference beads blue 1 μm BB Fluoresbrite Microspheres (17458; Polysciences)
Reference beads blue 0.5 μm BB Fluoresbrite Microspheres (18339; Polysciences)
Reference beads yellow-green 1 μm FluoSpheres (F-13081, Lot 03B2-1; ThermoFisher Scientific)
Reference beads yellow-green 0.5 μm FluoSpheres (F-8813, Lot. 69B3-1; ThermoFisher Scientific)
Laser alignment beads blue 1 μm FluoSpheres (F-8815; ThermoFisher Scientific)
Laser alignment beads yellow-green 2 μm FluoSpheres (F-8827; ThermoFisher Scientific)
Laser alignment BD FACSuiteTM CS&T research beads (650621; Becton, Dickinson and Company)
Laser alignment BD CS&T RUO beads (661414; Becton, Dickinson and Company)
Flow cytometric samples of the mCMC were repeatedly measured over months without changing community patterns. Flow
cytometric distributions were veriyfied by spiked beads of the size of 0.5 μm and 1 μm in each sample measured. Position of cells in
the cell gate and the gate template was mandatory.
Randomization not applicable.

The mCMCs were always constructed to known proportions: please see Supplementary Table 2 to Supplementary Table 6.
Blinding not applicable
Did the study involve field work? Yes No
Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,
system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
Materials & experimental systems Methods

n/a Involved in the study n/a Involved in the study
Antibodies ChIP-seq
Eukaryotic cell lines Flow cytometry
Palaeontology MRI-based neuroimaging
Animals and other organisms
Human research participants
Clinical data
Flow Cytometry
Plots
Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
October 2018
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
3
Methodology

Sample preparation lab cultures and wastewater types
Instrument BD Influx v7 Cell Sorter (BD, see Equipment Setup)

MoFlo Legacy Cell Sorter (Beckman Coulter, see Equipment Setup)
CyFlowSpace analyzer (Sysmex-Partec, see Equipment Setup)
BD FACSVerse analyzer (BD, see Equipment Setup)
BD Accuri C6+ analyzer (BD, see Equipment Setup)
Software FlowJoTM 10.0.8.r1 (FlowJo, Tree Star / BD)

R package flowCybar: http://bioconductor.org/packages/release/bioc/html/flowCyBar.html
R package flowCHIC: http://www.bioconductor.org/packages/release/bioc/html/flowCHIC.html
Cell population abundance We measured 50.000 cell for pure strains and 200.000 cells for artificial and natural communities within the respective cell gates.
Gating strategy Cells were measured outside of beads and noise and marked with a cell gate. For the beads a bead template was established
which was used for cytometer alignment per day and each sample. Upcoming subpopulations/subcommunities were marked by
an ellipsoid gate. Non-overlapping gates were created for each microbial community 2D-plot generating the gate template.
About 80% of all cells should be part of the gate template. The gate template was used to determine abundances of cells in the
gates of the gate template (see Supplementary Table 2 to Supplementary Table 6).
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.
October 2018

Nature Protocols 2020

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Nature Protocols 2020

Uploaded by

Copyright:

Available Formats

PROTOCOL EXTENSION

Bacterial mock communities as standards for

This protocol is an extension to Nat. Protoc. 8, 190–202 (2013): https://doi.org/10.1038/nprot.2012.149

Development of the protocol

2788 NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot

Application of the method

NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot 2789

Comparison with other methods

2790 NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot

Expertise needed to implement the protocol

NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot 2791

Strain ﬁxation and staining

Proportions of strains in either liquid or plate mCMCs

Gate setting for the liquid mCMC

Gate setting for the plate mCMC

The gate template

2792 NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot

100 L9 L11 L12

NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot 2793

Cytometric analysis and cell sorting for downstreaming applications

2794 NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot

d Liquid Plate Liquid Plate

L1 L2 L3 L4 L5 L6 L7 L9L11 A B C P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 L1 L2 L3 L4 L5 L6 L7 L9 L11 A B C P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11

NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot 2795

2796 NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot

DAPI fluorescence (log)

NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot 2797

● S. rhizophila DSM 14405 (DSMZ; Leibniz Institute)

● E. coli DSM 4230 (DSMZ; Leibniz Institute)

● Ethanol (Sigma-Aldrich, CAS no. 64-17-5)

● Potassium phosphate monobasic (KH PO ; Sigma-Aldrich, CAS no. 7778-77-0)

● EDTA (Thermo Fisher Scientiﬁc, cat. no. 17892)

● Agarose for gel electrophoresis (Sigma-Aldrich, CAS no. 9012-36-6)

● Immersion oil Type-F (Olympus)

● Citric acid (Sigma-Aldrich, CAS no. 77-92-9)

● Tween-20 (Sigma Aldrich, CAS no. 9005-64-5)

handling the solution.

gloves when handling the solution.

● BD FACSFlow buffer (BD, cat. no. 342003)

CRITICAL Always re-suspend the

● Chelex 100 sodium form (Sigma-Aldrich, CAS no. 11139-85-8)

● Molecular marker (Qiagen, cat. no. 239045)

Forward 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ (Euroﬁns Scientiﬁc)

2798 NATURE PROTOCOLS | VOL 15 | SEPTEMBER 2020 | 2788–2812 | www.nature.com/nprot

PCR2: Six-nucleotides-barcoded mother primers (Euroﬁns Scientiﬁc)

● Phusion GC solution (New England Biolabs, cat. no. B0519S)

● Phusion High-Fidelity Polymerase (New England Biolabs, cat. no. M0530S)

● Agencourt AMPure beads XP-Kit (Beckman Coulter, cat. no. A63880)

● dsDNA HS Assay Kit (Thermo Fisher Scientiﬁc, cat. no. Q32851)

● Ethanol 70% in bidistilled H O (vol/vol)

● Cell Trics (50 µm; Sysmex-Partec)

● Ultraspec III (Amersham Biosciences Europe)

● Microcentrifuge (Heraeus Fresco 21 Centrifuge, Thermo Fisher Scientiﬁc)

● Vortex (VF2, Janke Kunkel IKA Labortechnik)

● BD Inﬂux v7 Cell Sorter (BD; see ‘Equipment setup’ section)

● MoFlo Legacy Cell Sorter (Beckman Coulter; see ‘Equipment setup’)

● CyFlowSpace analyzer (Sysmex-Partec, see ‘Equipment setup’ section)

● BD FACSVerse analyzer (BD, see ‘Equipment setup’ section)

● BD Accuri C6+ analyzer (BD, see ‘Equipment setup’ section)

● PTC-200 Thermal Cycler (MJ Research)

● PCR machine (S1000 Thermal cycler, Biorad)

● Magnetic separation rack (magnetic Stand-96, Thermo Fisher Scientiﬁc)