Evaluation of volume-based flow cytometry as a potential primary method for quantification of bacterial reference material.

Bacterial reference materials (RMs) play a crucial role in many analytical processes of microbiological detection. Currently, bacteria are typically counted using the traditional plate-based approach, which results in a higher uncertainty of bacterial RMs unfortunately. Therefore, novel methods are urgently required for the value assignment of RMs in the field of microbiology to derive measurement traceability and accuracy. A potential primary method for microbiological quantification based on flow cytometry (FCM) is described in this study using Escherichia coli O157 (E. coli O157) as an example. The proposed method was applied to determine the number of viable E. coli O157 cells in the RMs with a result of (5.48 ± 0.27) × 108 cells mL-1, which was in good agreement with the result obtained using the plate-based method (En = 0.47). Additionally, this method could be entirely described and understood by equations, and provides formal traceability to the SI for counts of viable bacterial cells, while the associated relative expanded uncertainty (4.93%, k = 2) was significantly lower in comparison to the plate-based method. Therefore, the FCM-based method might be a potential primary method for characterizing bacterial RMs. To our knowledge, this is the first description of FCM as a potential primary method for accurate and traceable quantification of viable bacterial cells with a comprehensive uncertainty statement in microbiological metrology.