Measurement quality metrics to improve absolute microbial cell counting

Published

August 18, 2025

Author(s)

Kirsten Parratt, David Newton, Joy Dunkers, Jennifer Dootz, Monique Hunter, Alshae Logan, Laura Pierce, Sumona Sarkar, Stephanie Servetas, Nancy Lin

Abstract

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.

Citation

Frontiers in Microbiology

Volume

16

Pub Type

Journals

Download Paper

https://doi.org/10.3389/fmicb.2025.1631377

Local Download

Citation

Parratt, K. , Newton, D. , Dunkers, J. , Dootz, J. , Hunter, M. , Logan, A. , Pierce, L. , Sarkar, S. , Servetas, S. and Lin, N. (2025), Measurement quality metrics to improve absolute microbial cell counting, Frontiers in Microbiology, [online], https://doi.org/10.3389/fmicb.2025.1631377, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=958381 (Accessed May 30, 2026)

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