Enumeration of probiotic strains: Review of culture-dependent and alternative techniques to quantify viable bacteria

Abstract

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Standard culture techniques are commonly used to quantify probiotic strains, but cell culture only measures replicating cells. In response to the stresses of processing and formulation, some fraction of the live probiotic microbes may enter a viable but non-culturable state (VBNC) in which they are dormant but metabolically active. These microbes are capable of replicating once acclimated to a more hospitable host environment. An operating definition of live probiotic bacteria that includes this range of metabolic states is needed for reliable enumeration. Alternative methods, such as fluorescent in situ hybridization (FISH), nucleic acid amplification techniques such as real-time quantitative PCR (RT-qPCR or qPCR), reverse transcriptase (RT-PCR), propidium monoazide-PCR, and cell sorting techniques such as flow cytometry (FC)/fluorescent activated cell sorting (FACS) offer the potential to enumerate both culturable and VBNC bacteria. Modern cell sorting techniques have the power to determine probiotic strain abundance and metabolic activity with rapid throughput. Techniques such as visual imaging, cell culture, and cell sorting, could be used in combination to quantify the proportion of viable microbes in various metabolic states. Consensus on an operational definition of viability and systematic efforts to validate these alternative techniques ultimately will strengthen the accuracy and reliability of probiotic strain enumeration. • Probiotics are live microorganisms that confer a benefit on the host. • Live probiotic bacteria can exist in a viable but non-culturable state (VBNC). • Cell culture estimates numbers of replicating strains but not those in the VBNC state. • An operating definition of live probiotic bacteria is needed. • Culture-independent techniques can enumerate viable probiotic strains.