MICROBIAL CYTOMETRIC MOCK COMMUNITIES AND USE THEREOF AS STANDARD IN FLOW CYTOMETRY

Abstract

The present invention is directed to a microbial Cytometric Mock Community for use in flow cytometric analysis, the microbial Cytometric Mock Community comprising or consisting of cells of at least three different microbial species in a pre-defined ratio, wherein the at least three different microbial species are selected such that, when measured using flow cytometry, the specific gate pattern of each microbial species differs significantly from the specific gate pattern of the other microbial species of the microbial Cytometric Mock Community, preferably the at least three different microbial species differ in relative DNA content, relative genomic GC-content, relative cell size, Gram +/− affiliation and/or capacity to form spores. The microbial Cytometric Mock Community shall serve as standardization means that will help ecologists, microbiologists, molecular biologists and flow cytometrists to work on a standardized basis to allow comparison and exchange of data.

Claims

1. Microbial Cytometric Mock Community for use in flow cytometric analysis, the microbial Cytometric Mock Community comprising or consisting of cells of at least three different microbial species in a pre-defined ratio, wherein the at least three different microbial species are selected such that, when measured using flow cytometry, the specific gate pattern of each microbial species differs significantly from the specific gate pattern of the other microbial species of the microbial Cytometric Mock Community, preferably the at least three different microbial species differ in overall DNA content, relative genomic GC-content, average cell size, Gram +/− affiliation and/or capacity to form spores.

2. Microbial Cytometric Mock Community of claim 1, wherein the at least three different microbial species comprise or consist of species derived from archaea, bacteria, fungi, protozoa and algae, preferably derived from bacterial species.

3. Microbial Cytometric Mock Community according to claim 1, wherein the cells of the at least three different microbial species are derived from cultures each being in stationary state.

4. Microbial Cytometric Mock Community according to claim 1, wherein the cells of the at least three different microbial species have been fixated and, optionally, stained with nucleic acid specific fluorescent dyes.

5. Microbial Cytometric Mock Community according to claim 1, wherein the three different microbial species are selected from Kocuria rhizophila, Paenibacillus polymyxa, Stenotrophomonas rhizophila and Eschericha coli, preferably from the strains Kocuria rhizophila DSM 348, Paenibacillus polymyxa DSM 36, Stenotrophomonas rhizophila DSM 14405 and Eschericha coli DSM 4230.

6. Microbial Cytometric Mock Community according to claim 1, wherein the at least three different microbial species are Kocuria rhizophila DSM 348, Stenotrophomonas rhizophila DSM 14405 and at least one of Paenibacillus polymyxa DSM 36 and Eschericha coli DSM 4230.

7. Microbial Cytometric Mock Community according to claim 1, wherein the microorganisms of microbial Cytometric Mock Community comprise or consist of the three different microbial species are Kocuria rhizophila DSM 348, Stenotrophomonas rhizophila DSM 14405 and Paenibacillus polymyxa DSM 36.

8. Microbial Cytometric Mock Community according to claim 1, wherein the microorganisms of microbial Cytometric Mock Community comprise or consist of cells of four different microbial species, wherein said species are the strains Kocuria rhizophila DSM 348, Stenotrophomonas rhizophila DSM 14405, Paenibacillus polymyxa DSM 36 and Eschericha coli DSM 4230.

9. Microbial Cytometric Mock Community according to claim 1, wherein the microbial Cytometric Mock Community further comprises one or more types of beads suitable for flow cytometric measurement, preferably if more than one type of beads is present, the types of beads are selected such that their gates do not overlap with those of the cells when measured using flow cytometry.

10. A method of generating a gate template for standardization of flow cytometric analysis, the method comprising the steps of: providing the microbial Cytometric Mock Community of claim 1; fixating the microbial cells of the microbial Cytometric Mock Community; staining the microbial cells of the microbial Cytometric Mock Community; subjecting the stained microbial cells of the microbial Cytometric Mock Community to flow cytometric measurement; and defining the gates for the different microbial species of the microbial Cytometric Mock Community to form a gate template of the microbial Cytometric Mock Community.

11. A method of analysing a sample by standardized flow cytometry, the method comprising the steps of: providing a sample comprising microorganisms to be analysed by flow cytometry and the microbial Cytometric Mock Community of claim 1; processing the sample and the microbial Cytometric Mock Community in the same way, wherein processing encompasses fixation and staining of microbial cells; subjecting the processed sample and processed microbial Cytometric Mock Community to flow cytometric measurement; defining a gate template for standardisation by using the measurement data of the different microbial species of the microbial Cytometric Mock Community; and analysing the measurement data acquired for the sample in relation to the gate template defined for the microbial Cytometric Mock Community.

12. A kit comprising the microbial Cytometric Mock Community and a manual for performing the method of claim 10.

13. Use of the microbial Cytometric Mock Community of claim 1 in standardisation of flow cytometric measurement.

14. A kit comprising the microbial Cytometric Mock Community and a manual for performing the method of claim 11.

Description

FIGURES

[0062] FIG. 1: Creation of the gates for the microbial Cytometric Mock Communities. Left: Master gates (large geometric shape) which comprises 200,000 cells per measurement with gate templates (smaller gates). Two types of beads were included into each measurement (bead gates). Upper left: Master gate, gate template, and bead gates for the microbial Cytometric Mock Community cultivated on agar plates for 72 h. Lower left: Master gate, gate template, and bead gates for the microbial Cytometric Mock Community cultivated in liquid medium for 24 h. Right: Analyzed microbial Cytometric Mock Communities. Upper right: strains were cultivated on agar plates for 72 h, sampled, fixated, stained, and mixed at proportions 19:1:80 and measured cytometrically: Kocuria rhizophila DSM 348 (gates P4, P5, P6), Stenotrophomonas rhizophila DSM 14405 (gates P1, P2, P3), Paenibacillus polymyxa DSM 36 (gates P7, P8, P9, P10, P11). Lower right, strains that were cultivated in liquid medium for 24 h sampled, fixated, stained, and mixed at proportions 8:1:28:3 and measured cytometrically: Kocuria rhizophila DSM 348 (gates L5, L6, L7), Stenotrophomonas rhizophila DSM 14405 (gates L1, L2), Paenibacillus polymyxa DSM 36 (gates L8, L9, L10, L11, L12), and Escherichia coli DSM 4230 (gates L3, L4).

[0063] FIG. 2: Flow cytometric patterns of the cells that were cultivated on agar plates for 72 h. Left: Paenibacillus polymyxa DSM 36 (gates P7, P8, P9, P10, P11). Middle left: Stenotrophomonas rhizophila DSM 14405 (gates P1, P2, P3). Middle right: Kocuria rhizophila DSM 348 (gates P4, P5, P6). Right: microbial Cytometric Mock Community ‘Agar plate’. Per pure culture 50,000 cells and per master gate 200,000 cells were measured. Two types of beads were included in each measurement. Cell gates were set according to the gate template in FIG. 1.

[0064] FIG. 3: Flow cytometric patterns of the cells that were cultivated in liquid medium for 24 h. Left: Paenibacillus polymyxa DSM 36 (gates L8, L9, L10, L11, L12). Middle left: Stenotrophomonas rhizophila DSM 14405 (gates L1, L2). Middle: Kocuria rhizophila DSM 348 (gates L5, L6, L7). Middle right: Escherichia coli DSM 4230 (gates L3, L4). Right: microbial Cytometric Mock Community ‘Liquid medium’. Per pure culture 50,000 cells and per master gate 200,000 cells were measured. Two types of beads were included in each measurement. Cell gates were set according to the gate template in FIG. 1.

[0065] FIG. 4: Flow cytometric patterns of the cells that were cultivated over time in liquid medium for 24 h. Samples were taken at 0 h, and after 2 h, 4 h, and 24 hours of cultivation. First row: Paenibacillus polymyxa DSM 36 (gates L8, L9, L10, L11, L12). Second row: Stenotrophomonas rhizophila DSM 14405 (gates L1, L2). Third row: Kocuria rhizophila DSM 348 (gates L5, L6, L7). Forth row: Escherichia coli DSM 4230 (gates L3, L4). Per master gate 50,000 cells were measured. Two types of beads were included in each measurement. Cell gates were set according to the gate template in FIG. 1.

[0066] FIG. 5: NMDS-plot for determination of dissimilarity of technical and biological replicates of two microbial Cytometric Mock Communities originating from an agar plate and liquid medium. Euclidian distance calculation was used. The technical replicates showed the highest similarity while the two microbial Cytometric Mock Communities were the most dissimilar ones. Left community) (open circle): microbial Cytometric Mock Community ‘Agar plate’; right communities: 2 (triangle up): three biological replicates of the cytometric Cytometric Mock Community ‘Liquid medium’ and measured after 3 days storage at −20° C., 3 (straight cross): three biological replicates of the microbial Cytometric Mock Community ‘Liquid medium’ and measured after 2 months storage at −20 ° C., 4 (cross): technical replicates (of biological replicate nr. 2) of the microbial Cytometric Mock Community ‘Liquid medium’ measured after 2 months storage at −20 ° C., 5 (rhombus): technical replicates (of biological replicate nr. 3) of the microbial Cytometric Mock Community ‘Liquid medium’ measured after 2 months storage at −20 ° C., 6 (triangle down): technical replicates (of biological replicate nr. 1) of the microbial Cytometric Mock Community ‘Liquid medium’ measured after 2 months storage at −20 ° C., 7 (rectangle with cross): first repetitive measurement of 6 and 8, (star): second repetitive measurement of 6.

[0067] FIG. 6: Microbial Cytometric Mock Community ‘Liquid medium’ measured with a 355 nm laser at a constant laser power of 100 mW and with a 488 nm laser with decreasing laser power: A: 400 mW, B: 200 mW, C: 100 mW, D: 50 mW. The dotplot in E represents the microbial Cytometric Mock Community measured at 50 mW at an increased gain value for the FSC-PMT.

[0068] FIG. 7: Microbial Cytometric Mock Communities ‘Liquid medium’ analysed with DAPI that were created out of different proportions of the four strains from liquid culture, cultivated in liquid medium for 24 h. The strains were separately fixed, stained with DAPI and mixed in different proportions and measured. Per master gate 200,000 cells were measured. The proportions are given for Paenibacillus polymyxa DSM 36 (gates L8, L9, L10, L11, L12), Stenotrophomonas rhizophila DSM 14405 (gates L1, L2), Kocuria rhizophila DSM 348 (gates L5, L6, L7), Escherichia coli DSM 4230 (gates L3, L4), respectively. A) 70:2.5:20:7.5; B) 70:2.5:12.5:15; C) 81.4:2.8:14.4:1.4; D) 97.5:0.5:1.5:0.5; E) 45:5:15:35; F) 45:0.5:15:39.5.

[0069] FIG. 8: Microbial Cytometric Mock Communities ‘Agar plate’ analysed with DAPI that were created out of different proportions of the three strains from agar plate culture, cultivated for 72 h. The strains were separately fixed, stained with DAPI and mixed in different proportions and measured. Per master gate 200,000 cells were measured. The proportion are given for Paenibacillus polymyxa DSM 36 (gates P7, P8, P9, P10, P11) Stenotrophomonas rhizophila DSM 14405 (gates P1, P2, P3), Kocuria rhizophila DSM 348 (gates P4, P5, P6), respectively. A) 80:1:19; B) 80:19:1; C) 80:15:5; D) 53.3:42.7:4; E) 92:0.25:7.75; F) 60:10:30.

[0070] FIG. 9: Microbial Cytometric Mock Community ‘Agar plate’ analysed with SYBR Green that was created of the three strains from agar plate culture, cultivated for 72 h. The strains were separately fixed, stained with SYBR Green and mixed in the proportion of 33:33:33 and measured. Per pure culture 50,000 cells and per master gate 200,000 cells were measured. Left: Paenibacillus polymyxa DSM 36. Middle left: Stenotrophomonas rhizophila DSM 14405. Middle right: Kocuria rhizophila DSM 348. Right: microbial Cytometric Mock Community ‘Agar plate’. Two types of beads were included in each measurement.

[0071] FIG. 10: Microbial Cytometric Mock Community ‘Liquid medium’ analysed with SYBR Green that was created of the four strains from liquid medium culture, cultivated for 24 h. The strains were separately fixed, stained with SYBR Green and mixed in the proportion of 25:25:25:25 and measured. Per pure culture 50,000 cells and per master gate 200,000 cells were measured. Left: Paenibacillus polymyxa DSM 36. Middle left: Stenotrophomonas rhizophila DSM 14405. Middle: Kocuria rhizophila DSM 348. Middle right: Escherichia coli DSM 4230. Right: Cytometric Mock Community ‘Liquid medium’. Two types of beads were included in each measurement.

[0072] FIG. 11: Microbial Cytometric Mock Communities ‘Agar plate’ analysed with SYBR Green that were created out of different proportions of the three strains from agar plate culture, cultivated for 72 h. The strains were separately fixed, stained with SYBR Green and mixed in different proportions and measured. Per master gate 200,000 cells were measured. The proportions are given for Paenibacillus polymyxa DSM 36, Stenotrophomonas rhizophila DSM 14405, Kocuria rhizophila DSM 348. A) 55:31:14; B) 75:20:5; C) 40:50:10; D) 60:32.5:7.5; E) 60:37.5:2.5.

[0073] FIG. 12: Microbial Cytometric Mock Communities ‘Liquid medium’ analysed with SYBR Green that were created out of different proportions of the four strains from liquid culture, cultivated in liquid medium for 24 h. The strains were separately fixed, stained with SYBR Green and mixed in different proportions and measured. Per master gate 200,000 cells were measured. The proportions are given for Paenibacillus polymyxa DSM 36, Stenotrophomonas rhizophila DSM 14405, Kocuria rhizophila DSM 348, Escherichia coli DSM 4230. A) 50:15:15:20; B) 75:11:5:9; C) 40:12.5:12.5:35; D) 64.5:7.5:18:10; E) 75:2.5:10.5:12.

EXAMPLES

Results

Cultivation of Strains for the Microbial Cytometric Mock Community

[0074] The four strains Kocuria rhizophila DSM 348, Paenibacillus polymyxa DSM 36, Stenotrophomonas rhizophila DSM 14405, and Escherichia coli DSM 4230 were chosen to construct two different microbial Cytometric Mock Communities. For the first microbial Cytometric Mock Community, the strains were independently cultivated on LB agar plates for 72 hours. For the second microbial Cytometric Mock Community the respective strains were independently cultivated in liquid LB medium by taking one colony from agar plates (after 72 h) and its pre-cultivation for 24 h in liquid medium. The main cultures were started by inoculation of 1 ml (OD700 nm d=0.05) of the pre-culture and grown for another 24 h (cf. methods). The final four stationary state cultures served to create the microbial Cytometric Mock Community from liquid medium.

Creation of the Gate Templates

[0075] Stationary state liquid cultures and agar plate cultures were used to ensure stable populations states which are represented by discrete cytometric population patterns. These patterns are not homogeneous. DAPI/FSC pattern of a population describe cell size related cell characteristics and numbers of chromosomes per cell. A bacterial cell usually has one, sometimes also two or three chromosomes of different sizes and sequences. In addition, depending on states in the growth cycle bacteria can have many copies of a chromosome. The relative numbers of chromosomes per cell can be detected by DAPI staining. Therefore, a DAPI/FSC dotplot mirrors the heterogeneity of a population with regard to cell size and chromosome number of cells that cluster in different subpopulations. The position of cell clusters in a histogram and the numbers of clusters are strain specific and depend frequently on growth stages. All upcoming subpopulations can be marked by gates (FIG. 1). This fact is used to create strain specific gate templates within a master gate that comprises all gates (FIG. 1). Spiked beads of different types serve as control for the stable position of the master gate and the gate templates (FIG. 1, two gates outside the master gate).

[0076] To create a gate template for a microbial Cytometric Mock Community it is advisable to choose cells from relatively stable growth stages. For our two microbial Cytometric Mock Communities we used, as a first growth stage, the 72 h grown agar plate cultures and, as a second growth stage, the 24 h grown stationary state states liquid cultures. The following numbers of gates were defined for the four strains and the two growth stages, respectively: Kocuria rhizophila DSM 348 (3,3), Paenibacillus polymyxa DSM 36 (5,5), Stenotrophomonas rhizophila DSM 14405 (3,2), and Escherichia coli DSM 4230 (0,2). Gates of Escherichia coli DSM 4230 were found to overlap with gates from Paenibacillus polymyxa DSM 36 when cultivated on agar plates, therefore, we excluded this strain from the agar plate microbial Cytometric Mock Community.

Cytometric Patterns of Cells From Agar Plates

[0077] Cells of each strain were harvested from LB agar plates after 72 h, fixated and stained with DAPI and measured by flow cytometry. The strain specific patterns are shown in FIG. 2. The microbial Cytometric Mock Community ‘Agar Plate’ is created by mixing cells at proportions 19:1:80 and measuring them cytometrically: Kocuria rhizophila DSM 348 (gates P4, P5, P6), Stenotrophomonas rhizophila DSM 14405 (gates P1, P2, P3), and Paenibacillus polymyxa DSM 36 (gates P7, P8, P9, P10, P11).

[0078] Following the mentioned protocol, the agar plate cultures produce cell material for as much as 100 calibrations. The fixated cells must be stored at −20° C.

Cytometric Patterns of Cells From Liquid Media

[0079] Cells of each strain were harvested from 24 h grown stationary state liquid cultures, fixated and stained with DAPI and measured by flow cytometry. The specific patterns are shown in FIG. 3. The microbial Cytometric Mock Community ‘Liquid Medium’ is created by mixing cells at proportions 8:1:28:3 and measured cytometrically: Kocuria rhizophila DSM 348 (gates L5, L6, L7), Paenibacillus polymyxa DSM 36 (gates L8, L9, L10, L11, L12), Stenotrophomonas rhizophila DSM 14405 (gates L1, L2), and Escherichia coli DSM 4230 (gates L3, L4).

[0080] We also followed the different growth stages of the four strains in liquid culture after 0 h, 2 h, 4 h, and 24 h (FIG. 4). The cell cycle of a pure strain, i.e. increase in chromosome copy numbers per cell due to DNA replication or decrease due to cell division, can be observed by the distribution of cells within strain specific gates which are in this study the gates L8, L9, L10, L11, L12 for Paenibacillus polymyxa DSM 36, the gates L1, L2 for Stenotrophomonas rhizophila DSM 14405, the gates L5, L6, L7 for Kocuria rhizophila DSM 348, and the gates L3, L4 for Escherichia coli DSM 4230. The four strains were grown independently on liquid LB medium as biological triplicates and were inoculated with stationary state cultures, respectively. All strains reached the stationary state after 24 h where the flow cytometric population pattern of the inoculum at 0 h was identical to the pattern reached after 24 h. During exponential growth the cells did contain generally more DNA and did not cluster to clearly separated subpopulations.

[0081] Following the mentioned protocol, one batch of cultures produces cell material for as much as 100 calibrations. The fixated cells must be stored at −20° C.

Intrinsic Variation of Biological and Technical Samples in Flow Cytometric Patterns

[0082] To ensure the quality and the reliability of the data, biological as well as technical replicates of the two microbial Cytometric mock Communities from the agar plates and from the liquid culture were generated and cytometrically measured. The two respective main gate templates (see above) were used to evaluate the triplicate measurements by using the flowCHIC (https://www.bioconductor.org/packages/release/bioc/html/flowCHIC.html). The degree of deviation between the cytometrically measured biological and technical replicates was determined. The deviations between all technical samples showed extremely low Euclidian distance values. In contrast, the deviation between the microbial Cytometric Mock Community from the ‘agar plates’ and all samples from the ‘liquid medium’ was high according the Euclidian distance values (FIG. 5).

Influence of the Laser Power on the Flow Cytometric Fingerprints

[0083] Flow cytometers are not only equipped with different laser types and wavelengths, the power of the lasers can also be different. Increasing laser power certainly influences the fluorescence intensity values of a cell by creating higher photon numbers. Low-cost flow cytometers are often equipped with low-cost low-power lasers, therefore, we wanted to test if low-power lasers resolve the scatter of microbial Cytometric Mock Community members accurately. While the 355 nm laser line of the Influx was a fixed line with a power of 100 mW, the 488 nm laser was equipped with an adjustable power option which was used to analyze the microbial Cytometric Mock Community of the liquid culture at 400 mW, 200 mW, 100 mW, and 50 mW (FIG. 6; A, B, C, D, respectively). A microbial Cytometric Mock Community was used that was stored at −20° C. We found the microbial Cytometric Mock Community to shift by about 1.2 magnitudes to lower values in the forward scatter channel in comparison to the gate template (FIG. 6). Although the distribution and resolution of the microbial Cytometric Mock Community members did not change it became clear that the laser power certainly influenced the position of the microbial Cytometric Mock Community in the histogram. We usually recommend placing the microbial Cytometric Mock Community into the middle of the histogram to enable the measurement of both smaller and larger cells in follow-up experiments. Therefore, we tested, if increasing the gain of the FSC-PMT could replace the microbial Cytometric Mock Community into its former position. This was possible, and thus, even low-power lasers guaranty a high resolution of the members of a microbial Cytometric Mock Community (FIG. 6 E).

Influence of Different Proportions of Strains From Liquid Culture on Microbial Cytometric Mock Community Pattern Using DAPI

[0084] We analysed different proportions of strains within the microbial Cytometric Mock Community when stained with DAPI in order to test if other proportions might also be useful or might distort the structure of the microbial Cytometric Mock Community. All four strains were obtained from liquid cultures, respectively, and cultivated in liquid medium for 24 h. The strains were separately fixed, stained with DAPI and mixed in different proportions and measured. Per master gate 200,000 cells were measured. The proportions are given for Paenibacillus polymyxa DSM 36 (gates L8, L9, L10, L11, L12), Stenotrophomonas rhizophila DSM 14405 (gates L1, L2), Kocuria rhizophila DSM 348 (gates L5, L6, L7), Escherichia coli DSM 4230 (gates L3, L4), respectively. A) 70:2.5:20:7.5; B) 70:2.5:12.5:15; C) 81.4:2.8:14.4:1.4; D) 97.5:0.5:1.5:0.5; E) 45:5:15:35; F) 45:0.5:15:39.5. All proportions show well resolved microbial Cytometric Mock Community patterns and can all be used for cytometric calibration (FIG. 7).

Influence of Different Proportions of Strains From Plate Culture on Microbial Cytometric Mock Community Pattern Using DAPI

[0085] We analysed different proportions of strains within the microbial Cytometric Mock Community when stained with DAPI in order to test if other proportions might also be useful or might distort the structure of the microbial Cytometric Mock Community. All three strains were obtained from plate cultures, respectively, and cultivated on plates for 72 h. The strains were separately fixed, stained with DAPI and mixed in different proportions and measured. Per master gate 200,000 cells were measured. The proportions are given for Paenibacillus polymyxa DSM 36 (gates P7, P8, P9, P10, P11) Stenotrophomonas rhizophila DSM 14405 (gates P1, P2, P3), Kocuria rhizophila DSM 348 (gates P4, P5, P6), respectively. A) 80:1:19; B) 80:19:1; C) 80:15:5; D) 53.3:42.7:4; E) 92:0.25:7.75; F) 60:10:30 (FIG. 8).

Influence of SYBR Green Staining on the Structure of the Microbial Cytometric Mock Community Pattern Originating From Plate Cultures

[0086] Many common flow cytometers are not equipped with an UV laser which is necessary to excite DAPI: Therefore, we tested the nucleic acid dye SYBR Green (excitation 488 nm) for its usefulness to stain the microbial Cytometric Mock Community and create well resolved patterns useful for calibration of such cytometers. We found that the patterns were not as highly resolved as was possible with DAPI, but nevertheless high enough to be useful as a microbial Cytometric Mock Community. Three strains, originating from agar plate culture, were cultivated for 72 h: Paenibacillus polymyxa DSM 36, Stenotrophomonas rhizophila DSM 14405, and Kocuria rhizophila DSM 348. The strains were separately fixed, stained with SYBR Green and mixed in the proportion of 33:33:33 and measured as ‘Agar plate’ microbial Cytometric Mock Community. Per pure culture 50,000 cells and per master gate 200,000 cells were measured and two types of beads were included in each measurement (FIG. 9).

Influence of SYBR Green Staining on the Structure of the Microbial Cytometric Mock Community Pattern Originating From Liquid Cultures

[0087] We also tested the nucleic acid dye SYBR Green (excitation 488 nm) for its usefulness to stain the microbial Cytometric Mock Community and create well resolved patterns useful for calibration of such cytometers from liquid cultures. We found again that the patterns were not as highly resolved as was possible with DAPI, but nevertheless high enough to be useful as a microbial microbial Cytometric Mock Community. Four strains, originating from agar plate culture, were cultivated for 24 h: Paenibacillus polymyxa DSM 36, Stenotrophomonas rhizophila ; DSM 14405, Escherichia coli DSM 4230 and Kocuria rhizophila DSM 348. The strains were separately fixed, mixed in the proportion of 25:25:25:25, stained with SYBR Green and measured as ‘Liquid medium’ microbial Cytometric Mock Community. Per pure culture 50,000 cells and per master gate 200,000 cells were measured and two types of beads were included in each measurement (FIG. 10).

Influence of Different Proportions of Strains from Agar Plate Culture on Microbial Cytometric Mock Community Pattern Using SYBR Green

[0088] We also analysed different proportions of strains within the microbial Cytometric Mock Community when stained with SYBR Green in order to test if other proportions might also be useful or might distort the structure of the microbial Cytometric Mock Community. All three strains were obtained from agar plate cultures, respectively, and cultivated on agar plates for 72 h. The strains were separately fixed, mixed in different proportions, stained with SYBR Green and measured. Per master gate 200,000 cells were measured. The proportions are given for Paenibacillus polymyxa DSM 36, Stenotrophomonas rhizophila DSM 14405, Kocuria rhizophila DSM 348. A) 55:31:14; B) 75:20:5; C) 40:50:10; D) 60:32.5:7.5; E) 60:37.5:2.5. All proportions show well resolved microbial Cytometric Mock Community patterns (although not as well resolved as with DAPI) and can all be used for cytometric calibration (FIG. 11).

Influence of Different Proportions of Strains from Liquid Culture on microbial Cytometric Mock Community Pattern Using SYBR Green

[0089] We analysed different proportions of strains within the microbial Cytometric Mock Community when stained with SYBR Green in order to test if other proportions might also be useful or might distort the structure of the microbial Cytometric Mock Community. All four strains were obtained from liquid cultures, respectively, and cultivated in liquid culture for 24 h. The strains were separately fixed, mixed in different proportions, stained with SYBR Green and measured. Per master gate 200,000 cells were measured. The proportions are given for Paenibacillus polymyxa DSM 36, Stenotrophomonas rhizophila DSM 14405, Kocuria rhizophila DSM 348, Escherichia coli DSM4230. A) 50:15:15:20; B) 75:11:5:9; C) 40:12.5:12.5:35; D) 64.5:7.5:18:10; E) 75:2.5:10.5:12. All proportions show well resolved microbial Cytometric Mock Community patterns (although not as well resolved as with DAPI) and can all be used for cytometric calibration (FIG. 12).

Material and Methods

Strains and Cultivation

[0090] The Cytometric Mock Community was constructed using following strains from the DSMZ: Kocuria rhizophila DSM 348, Paenibacillus polymyxa DSM 36, Stenotrophomonas rhizophila DSM 14405, Escherichia coli DSM 4230. The strains were handled following the DSMZ's recommendations, placed on LB-agar plates (Lysogeny Broth, Yeast extract 5 g/L, NaCl 5 g/L, Tryptone 10 g/L, pH 7.0, Agar 20 g/L, Carl ROTH GmbH, Karlsruhe, Germany) at 30° C. for 72 h. Afterwards a colony served as inoculum for a 100 mL liquid flask containing 20 mL of LB medium which was grown at 30° C. for 24 h at 150 rpm. This pre-cultivation step was done in triplicates and OD measured (d.sub.λ700nm=0.5 cm, Ultrospec III Amersham Biosciences Europe). Following, triplicate 500 mL flasks, filled with 100 mL of LB medium, were inoculated to an OD=0.05 (d.sub.λ700nm=0.5 cm) with cells of the pre-culture and grown at 30° C. for 24 h at 150 rpm.

Cell Sampling and Fixation

[0091] Of the cell suspension 5 to 8 mL were taken, centrifuged (3.200 g, 10 min, 4° C.) and the supernatant discarded. The cells were washed in 3 ml phosphate buffered saline (PBS: 6 mM Na.sub.2HPO.sub.4, 1.8 mM NaH.sub.2PO.sub.4, 145 mM NaCl in bi-destilled H.sub.2O, pH 7) once (3.200 g, 15 min, 4° C.) and the supernatant discarded. The cells were stabilized by adding 8 mL of para-formaldehyde solution (PFA, 2% in PBS) to the cell pellet for 30 min at room temperature. For a homogenized reaction, the pellet should be vortexed. After another centrifugation step (3.200 g, 15 min, 4° C., and discarding the supernatant), 8 mL of ethanol (70% in bi-distilled water) were added for fixation and the cell solution stored at −20° C. for two months maximum.

Cell Staining: DAPI

[0092] The OD (d.sub.λ700nm=0.5 cm) of the fixated cells was adjusted to 0.04 with PBS. Two ml of this solution were centrifuged (3.200 g, 15 min, 4° C.) and the supernatant discarded. The cell-pellet was resuspended in 1 mL of permeabilization buffer (0.1 M citric acid, 4.1 mM Tween 20, bi-destilled H.sub.2O) and incubated for 20 min at room temperature After a further centrifugation step the supernatant was discarded and the cells were resuspended in 2 ml 0.24 μM DNA-DAPI staining solution (4′,6-di-amidino-2-phenyl-indole, Sigma-Aldrich, St. Louis, USA) in Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer (289 mM Na.sub.2HPO.sub.4, 128 mM NaH.sub.2PO.sub.4 with bi-distilled H.sub.2O, pH 7) for subsequent staining overnight in the dark until flow cytometric measurement. Samples were filtered through 50 μm CellTrics® (Partec, Germany) prior to cytometric measurement. Fluorescence beads ((0.5 and 1 μm BB Fluoresbrite Microspheres (18339, 17458; Polysciences, Warrington, Pa., USA)) were added to the samples as internal standard. For measurements of single strains 50,000 and of microbial Cytometric Mock Communities 200,000 cells, respectively, were recorded.

Cell Staining: SYBR Green

[0093] The preparation of the cells for the staining was identical to the method above. In short, the fixated cells were adjusted to an OD (d.sub.λ700nm=0.5 cm) of 0.04 with PBS and 2 ml of this solution centrifuged (3.200 g, 10 min, 4° C.). The cells were pre-incubated for 4 min at 37° C., SYBR Green I (ThermoFisher Scientific, Waltham, Mass., USA) was added (final conc. 0.1×), and the cells were incubated at 37° C. for 20 min before measurement. Fluorescence beads (0.5 μm FluoSpheres carboxylate-modified microspheres, yellow-green fluorescent (505/515); F8813; and 1.0 μm FluoSpheres polystyrene microspheres, yellow-green fluorescent (505/515), F13081; ThermoFischer Sci.) were added to the samples as internal standard. For measurements of single strains 50,000 and of microbial Cytometric Mock Communities 200,000 cells, respectively, were recorded.

Flow Cytometric Analysis

[0094] Cytometric measurements were performed with a BD Influx v7 Sorter USB, (Becton, Dickinson and Company, Franklin Lakes, USA) equipped with a blue 488 nm Sapphire OPS laser (400 mW) and a 355 nm Genesis OPS laser (100 mW, both Coherent, Santa Clara, Calif., USA).

[0095] The 488 nm laser was used for analysis of forward scatter (FSC, 488/10), side scatter (SSC, trigger signal, 488/10), and the SYBR Green I fluorescence (530/40), while the 355 nm laser excited the DAPI fluorescence (460/50). Light was detected by Hamamatsu R3896 PMTs in C6270 sockets (Hamamatsu, 211 Hamamatsu City, Japan). The fluidic system was run at 33 psi with sample overpressure at 0.5 psi and a 70 μm nozzle. The sheath fluid consisted of FACSFlow buffer (BD) sample. Samples were analyzed at a speed of 2500 events s.sup.−1. Cytometric data were evaluated using FlowJo v10.0.8r1 with the Engine v3.04910 (FlowJo, LLC, Ashland, USA) and the R packages flowCyBar and flowCHIC (Bioconductor platform).

CONCLUSION

[0096] If we want to bring flow cytometry to the next level and help to shape and develop micro-ecology, health (microbiome) and biotechnology fields during the upcoming years, standardization is one of the mandatory steps to proceed to new levels of knowledge as it will allow creating standardized and comparable data between studies and labs. We are certain that standardization will help ecologists, microbiologists, molecular biologists and flow cytometrists to exchange hypothesis and increase scientific knowledge by working together and comparing data on a standardized basis. We are certain that the Microbial Cytometric Mock community allows the measurement of accurate population or community dynamics in a much better way than it is possible to date and will help to analyze dynamics of microbial communities in many applications such as environment, human and animal health or in biotechnology.