High-Throughput Viability Testing of Microbial Communities in a Probiotic Product Using Flow Cytometry
Abstract
:1. Introduction
2. Materials and Methods
2.1. Factors Group Australia Multi-Species Probiotic Product
2.2. Sample Processing
2.3. Enumeration and Sequencing of Viable Cells by the Plate Count Method
2.4. Generation of Dead Cell Control Populations for Flow Cytometric Analysis
2.5. Cell Viability Staining for Flow Cytometric Analysis
2.6. Flow Cytometric Data Acquisition and Analysis
2.7. Tolerance to Conditions Simulating Gastrointestinal (GI) Tract Passage
2.8. Statistical Analysis
3. Results
3.1. Determining Viable Cell Number Variability for Factors Group Australia Probiotic Capsules
3.2. Impact of Filtering out Prebiotic Fibres on the Viable Cell Counts of the Multi-Species Probiotic Product
3.3. Enumeration of Viable Cells in the Multi-Species Probiotic Product
3.4. Testing the Reproducibility of the Flow Cytometry Protocol
3.5. Comparison of Flow Cytometry and Plate Counting Methods for Assessing Viability following Simulated GI Tract Digestion
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Samples | (Mean ± SD) | |
---|---|---|
CFU/Capsule | Log10 CFU/Capsule | |
Capsule 1 | 3.4 ± 0.5 × 109 | 9.5 ± 0.09 |
Capsule 2 | 3.2 ± 0.1 × 109 | 9.5 ± 0.01 |
Capsule 3 | 3.1 ± 0.2 × 109 | 9.5 ± 0.02 |
Viable Cell Counts (Mean ± SD) | ||
---|---|---|
CFU/Capsule | Log10 CFU/Capsule | |
Filtrate cell count | 2.2 ± 0.1 × 109 | 9.3 ± 0.02 |
Capsule cell count (unfiltered) | 2.3 ± 0.1 × 109 | 9.4 ± 0.02 |
Viable Cell Counts (Mean ± SD) | ||
---|---|---|
Flow cytometry | 6.4 ± 0.4 × 109 cells/capsule | 9.8 ± 0.03 Log10 cells/capsule |
Plate count | 2.0 ± 0.1 × 109 CFU/capsule | 9.3 ± 0.03 Log10 CFU/capsule |
Samples | Viable Cell Counts (Mean ± SD) | |||
---|---|---|---|---|
Flow Cytometry | Plate Count | |||
Cells/Capsule | Log10 Cells/Capsule | CFU/Capsule | Log10 CFU/Capsule | |
Capsule 1 | 6.8 ± 0.2 × 109 | 9.8 ± 0.01 | 4.5 ± 0.5 × 107 | 7.6 ± 0.05 |
Capsule 2 | 6.1 ± 0.2 × 109 | 9.8 ± 0.01 | 5.1 ± 0.2 × 107 | 7.7 ± 0.02 |
Capsule 3 | 5.6 ± 0.1 × 109 | 9.7 ± 0.01 | 3.9 ± 0.3 × 107 | 7.6 ± 0.03 |
Capsule 4 | 7.1 ± 0.4 × 109 | 9.9 ± 0.02 | 4.3 ± 0.4 × 108 | 8.6 ± 0.04 |
Capsule 5 | 6.6 ± 0.1 × 109 | 9.8 ± 0.01 | 6.3 ± 0.2 × 108 | 8.8 ± 0.02 |
Capsule 6 | 6.9 ± 0.2 × 109 | 9.8 ± 0.01 | 5.4 ± 0.5 × 108 | 8.7 ± 0.04 |
Exposure Time (Hours) | 0.3% Pepsin (pH 2.0) (Mean ± SD) | |||
---|---|---|---|---|
Flow Cytometry | Plate Count | |||
Cells/Capsule | Log10 Cells/Capsule | CFU/Capsule | Log10 CFU/Capsule | |
0 | 8.5 ± 0.2 × 109 | 9.9 ± 0.01 | 9.7 ± 0.3 × 108 | 8.8 ± 0.02 |
1.5 | 3.9 ± 0.2 × 109 | 9.6 ± 0.03 | 9.3 ± 0.3 × 107 | 7.9 ± 0.01 |
3 | 3.9 ± 0.5 × 109 | 9.6 ± 0.06 | 9.1 ± 0.8 × 107 | 7.9 ± 0.04 |
Exposure Time (Hours) | 0.3% Bile Salts (pH 7.0) (Mean ± SD) | |||
---|---|---|---|---|
Flow Cytometry | Plate Count | |||
Cells/Capsule | Log10 Cells/Capsule | CFU/Capsule | Log10 CFU/Capsule | |
0 | 8.1 ± 0.3 × 109 | 9.9 ± 0.02 | 4.4 ± 0.6 × 108 | 8.6 ± 0.07 |
1.5 | 4.6 ± 0.3 × 109 | 9.7 ± 0.02 | 1.9 ± 0.2 × 107 | 7.8 ± 0.06 |
3 | 4.8 ± 0.2 × 109 | 9.7 ± 0.02 | 1.6 ± 0.1 × 107 | 7.7 ± 0.03 |
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Pereira, J.V.; Gamage, H.K.A.H.; Cain, A.K.; Hayes, E.; Paulsen, I.T.; Tetu, S.G. High-Throughput Viability Testing of Microbial Communities in a Probiotic Product Using Flow Cytometry. Appl. Microbiol. 2023, 3, 1068-1082. https://doi.org/10.3390/applmicrobiol3030074
Pereira JV, Gamage HKAH, Cain AK, Hayes E, Paulsen IT, Tetu SG. High-Throughput Viability Testing of Microbial Communities in a Probiotic Product Using Flow Cytometry. Applied Microbiology. 2023; 3(3):1068-1082. https://doi.org/10.3390/applmicrobiol3030074
Chicago/Turabian StylePereira, Joanna V., Hasinika K. A. H. Gamage, Amy K. Cain, Evan Hayes, Ian T. Paulsen, and Sasha G. Tetu. 2023. "High-Throughput Viability Testing of Microbial Communities in a Probiotic Product Using Flow Cytometry" Applied Microbiology 3, no. 3: 1068-1082. https://doi.org/10.3390/applmicrobiol3030074