Measurement quality metrics to improve absolute microbial cell counting
Total and viable microbial cell counts are increasingly important for applications including live biotherapeutic products, food safety, and probiotics. In microbiology, cells are quantified using methods such as colony forming unit (CFU), flow cytometry, and polymerase chain reaction (PCR), but diff...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2025-08-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1631377/full |
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| author | Kirsten Parratt David Newton Joy Dunkers Jennifer Dootz Monique Hunter Alshae' Logan-Jackson Laura Pierce Sumona Sarkar Stephanie L. Servetas Nancy J. Lin |
| author_facet | Kirsten Parratt David Newton Joy Dunkers Jennifer Dootz Monique Hunter Alshae' Logan-Jackson Laura Pierce Sumona Sarkar Stephanie L. Servetas Nancy J. Lin |
| author_sort | Kirsten Parratt |
| collection | DOAJ |
| description | Total and viable microbial cell counts are increasingly important for applications including live biotherapeutic products, food safety, and probiotics. In microbiology, cells are quantified using methods such as colony forming unit (CFU), flow cytometry, and polymerase chain reaction (PCR), but different methods measure different aspects of the cells (measurands), and results may not be directly comparable across methods. In the absence of a ground-truth reference material for cell count, one cannot quantify the accuracy of any cell counting method, which limits method performance assessments and comparisons. Herein, a modified analysis of cell counting methods based on the ISO 20391-2:2019 standard was developed and demonstrated for microbial cell samples diluted over a log-scale range of concentrations. Escherichia coli samples ranging in concentration from ~5 x 105 cells/mL to 2 x 107 cells/mL were quantified using CFU, Coulter principle, fluorescence flow cytometry, and impedance flow cytometry. Quality metrics modified from the ISO standard were calculated for each method and shown to be repeatable across replicate experiments. The quality metrics illustrate large differences in proportionality and variability across methods, with total cell counts in good agreement and viable cell count having more variability. As the ISO standard is meant to guide fit-for-purpose method selection, interpretation of the results and quality metrics can drive method choice and optimization. The framework introduced here will help researchers select fit-for-purpose counting methods for quantification of microbial total and viable cells across a range of applications. |
| format | Article |
| id | doaj-art-244d976e71e2463a93c71b7a28452e27 |
| institution | Kabale University |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-244d976e71e2463a93c71b7a28452e272025-08-20T03:43:55ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-08-011610.3389/fmicb.2025.16313771631377Measurement quality metrics to improve absolute microbial cell countingKirsten Parratt0David Newton1Joy Dunkers2Jennifer Dootz3Monique Hunter4Alshae' Logan-Jackson5Laura Pierce6Sumona Sarkar7Stephanie L. Servetas8Nancy J. Lin9Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, United StatesStatistical Engineering Division, National Institute of Standards and Technology, Boulder, CO, United StatesBiosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, United StatesBiosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, United StatesBiosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, United StatesBiosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, United StatesBiosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, United StatesBiosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, United StatesBiosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, United StatesBiosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, United StatesTotal and viable microbial cell counts are increasingly important for applications including live biotherapeutic products, food safety, and probiotics. In microbiology, cells are quantified using methods such as colony forming unit (CFU), flow cytometry, and polymerase chain reaction (PCR), but different methods measure different aspects of the cells (measurands), and results may not be directly comparable across methods. In the absence of a ground-truth reference material for cell count, one cannot quantify the accuracy of any cell counting method, which limits method performance assessments and comparisons. Herein, a modified analysis of cell counting methods based on the ISO 20391-2:2019 standard was developed and demonstrated for microbial cell samples diluted over a log-scale range of concentrations. Escherichia coli samples ranging in concentration from ~5 x 105 cells/mL to 2 x 107 cells/mL were quantified using CFU, Coulter principle, fluorescence flow cytometry, and impedance flow cytometry. Quality metrics modified from the ISO standard were calculated for each method and shown to be repeatable across replicate experiments. The quality metrics illustrate large differences in proportionality and variability across methods, with total cell counts in good agreement and viable cell count having more variability. As the ISO standard is meant to guide fit-for-purpose method selection, interpretation of the results and quality metrics can drive method choice and optimization. The framework introduced here will help researchers select fit-for-purpose counting methods for quantification of microbial total and viable cells across a range of applications.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1631377/fullmeasurement quality metricsabsolute cell countmicrobial cell viabilityfluorescence flow cytometryimpedance flow cytometryCFU |
| spellingShingle | Kirsten Parratt David Newton Joy Dunkers Jennifer Dootz Monique Hunter Alshae' Logan-Jackson Laura Pierce Sumona Sarkar Stephanie L. Servetas Nancy J. Lin Measurement quality metrics to improve absolute microbial cell counting Frontiers in Microbiology measurement quality metrics absolute cell count microbial cell viability fluorescence flow cytometry impedance flow cytometry CFU |
| title | Measurement quality metrics to improve absolute microbial cell counting |
| title_full | Measurement quality metrics to improve absolute microbial cell counting |
| title_fullStr | Measurement quality metrics to improve absolute microbial cell counting |
| title_full_unstemmed | Measurement quality metrics to improve absolute microbial cell counting |
| title_short | Measurement quality metrics to improve absolute microbial cell counting |
| title_sort | measurement quality metrics to improve absolute microbial cell counting |
| topic | measurement quality metrics absolute cell count microbial cell viability fluorescence flow cytometry impedance flow cytometry CFU |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1631377/full |
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