Measurement quality metrics to improve absolute microbial cell counting.

Area of Science:

• Microbiology
• Biotechnology
• Analytical Chemistry

Background:

• Accurate microbial cell quantification is vital for live biotherapeutic products, food safety, and probiotics.
• Current methods like colony forming unit (CFU), flow cytometry, and polymerase chain reaction (PCR) measure different aspects of cells, leading to non-comparable results.
• The lack of a ground-truth reference material hinders the accuracy assessment of cell counting methods.

Purpose of the Study:

• To develop and demonstrate a modified analysis of microbial cell counting methods based on the ISO 20391-2:2019 standard.
• To assess the performance and comparability of different cell counting techniques for microbial samples.
• To provide a framework for selecting fit-for-purpose cell counting methods.

Main Methods:

• A modified ISO 20391-2:2019 standard analysis was applied to microbial cell samples.
• Escherichia coli samples were quantified using Colony Forming Unit (CFU), Coulter principle, fluorescence flow cytometry, and impedance flow cytometry.
• Quality metrics were calculated for each method to assess repeatability, proportionality, and variability.

Main Results:

• The developed quality metrics were repeatable across replicate experiments.
• Significant differences in proportionality and variability were observed across the tested cell counting methods.
• Total cell counts showed good agreement, while viable cell counts exhibited greater variability.

Conclusions:

• The introduced framework and quality metrics aid in understanding method performance for microbial cell quantification.
• Interpretation of results can guide the selection and optimization of fit-for-purpose counting methods.
• This approach supports researchers in choosing appropriate methods for diverse applications requiring microbial cell count accuracy.