Revisiting Strategies for Bacterial Enumeration: The Case for Viable but Nonculturable (VBNC) Cells in Supplement Products

Bacteria play an important role in both health and disease making the accuracy of methods used in their detection and quantification critical. Consistent with this, bacterial enumeration is an important technique used to determine the pathogenicity of foods/products as well as the potential for health benefits. The current preferred methods for bacterial enumeration include cultural detection and plate counting, despite not all bacterial species being culturable in a laboratory setting. For instance, fastidious bacteria, persister cells, and viable but nonculturable (VBNC) cells are examples where culturing might be difficult or impossible, thus rendering plate counting impractical. In this review, particular attention is given to the biology, historical context, and importance of VBNC cells in probiotics and supplement products. The different techniques used to enumerate bacteria within samples are explored for VBNC cells including plate counting, turbidimetrics, microscopy, flow cytometry, and digital polymerase chain reaction (dPCR). We review how advancements in dPCR have empowered the method to be sensitive to these non-culturable bacterial cells, and how the inclusion of quantitative PCR with viability dyes could be a practical solution. Given the significant human and environmental health impacts of bacteria, the importance of accurate enumeration and methodological improvement for both assessment and benchmarking is critical. Key Terms: Probiotics, Enumeration, Viable but Nonculturable, CFU, Digital PCR.