Development of an Automated Online Flow Cytometry Method to Quantify Cell Density and Fingerprint Bacterial Communities
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cultivation and Harvest of Bacterial Cells
2.2. NaCl/NaN3/EtOH Fixation
2.3. Paraformaldehyde (PFA)/EtOH Fixation for Standard Cytometric Analysis
2.4. DAPI Staining and Final Dilution for Standard Cytometric Analysis
2.5. Flow Cytometer
2.6. OC-300
2.7. Online Flow Cytometry Workflow for the Dilution, Fixation and Staining of Bacterial Cells
2.8. Cell Count Measurement
2.9. Stability of Measured Cell Concentration over Time Using the Online Flow Cytometry Workflow
2.10. Self-Cleaning Capabilities of the OC-300 Automation Unit between Measurements
2.11. Bioinformatics Evaluation
3. Results
3.1. Automated Dilution, Fixation, Staining and Analysis of Microbial Community Samples
3.2. Reliable Self-Cleaning Procedures of the OC-300
3.3. Reliability of Automatically Determined Cell Counts
4. Discussion
4.1. Development of an Automated Online Procedure to Obtain Fingerprints of Microbial Communities
4.2. Reliable Self-Cleaning Procedures of the OC-300
4.3. Reliability of Automatically Determined Cell Counts
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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MilliQ H2O | 100% FlowClean Cleaning Agent | 10% FlowClean Cleaning Agent | 1% FlowClean Cleaning Agent | ||
---|---|---|---|---|---|
P. polymyxa | Cell concentration in bacterial sample (cells/mL) | 5.14 × 108 ± 20.1% | 6.25 × 108 ± 25.7% | 7.02 × 108 ± 25.6% | 2.83 × 108 ± 21.5% |
Cell concentration in water sample (cells/mL) | 3.88 × 107 ± 18.4% | 4.89 × 107 ± 29.7% | 2.20 × 107 ± 24.7% | 1.08 × 107 ± 11.7% | |
Carryover percentage | 7.6 ± 2.3% | 7.83 ± 2.7% | 3.1 ± 0.4% | 3.8 ± 1.0% | |
K. rhizophila | Cell concentration in bacterial sample (cells/mL) | 4.99 × 108 ± 10.9% | 2.65 × 108 ± 10.0% | 4.82 × 108 ± 21.0% | 4.12 × 108 ± 11.4% |
Cell concentration in water sample (cells/mL) | 1.54 × 107 ± 23.4% | 8.39 × 106 ± 12.2% | 1.02 × 107 ± 21.6% | 4.37 × 106 ± 15.1% | |
Carryover percentage | 3.1 ± 3.1% | 3.2 ± 0.5% | 2.1 ± 0.8% | 1.1 ± 0.2% | |
S. rhizophila | Cell concentration in bacterial sample (cells/mL) | 1.25 × 109 ± 21.2% | 4.05 × 109 ± 9.1% | 1.51 × 109 ± 8.37% | 1.39 × 109 ± 10.5% |
Cell concentration in water sample (cells/mL) | 4.27 × 107 ± 6.3% | 9.66 × 107 ± 7.9% | 4.22 × 107 ± 18.5% | 3.85 × 107 ± 12.4% | |
Carryover percentage | 3.41 ± 0.6% | 2.4 ± 0.2% | 2.8 ± 0.5% | 2.9 ± 0.6% |
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López-Gálvez, J.; Schiessl, K.; Besmer, M.D.; Bruckmann, C.; Harms, H.; Müller, S. Development of an Automated Online Flow Cytometry Method to Quantify Cell Density and Fingerprint Bacterial Communities. Cells 2023, 12, 1559. https://doi.org/10.3390/cells12121559
López-Gálvez J, Schiessl K, Besmer MD, Bruckmann C, Harms H, Müller S. Development of an Automated Online Flow Cytometry Method to Quantify Cell Density and Fingerprint Bacterial Communities. Cells. 2023; 12(12):1559. https://doi.org/10.3390/cells12121559
Chicago/Turabian StyleLópez-Gálvez, Juan, Konstanze Schiessl, Michael D. Besmer, Carmen Bruckmann, Hauke Harms, and Susann Müller. 2023. "Development of an Automated Online Flow Cytometry Method to Quantify Cell Density and Fingerprint Bacterial Communities" Cells 12, no. 12: 1559. https://doi.org/10.3390/cells12121559