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.

Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids

“…However, these authors used this viability as a quality parameter, independent of the industrial matrix. The existence of similar subpopulations has also been depicted in microorganisms other than fungi (33)(34)(35). In analogy with those FCM strategies, it is of critical importance to take into account the number of viable conidia, even if these do not yet germinate at the time of sample assessment.…”

Germ-free sea bass Dicentrarchus labrax larval model: a valuable tool in the study of host-microbe interactions

“…Most prior studies working on gnotobiotic models relied solely on culture techniques, which are rather time consuming and may give false-negative results when dealing with slow-growing, or viable but non-culturable, organisms (Davis 2014). Therefore, non-culture based methods should be used additionally to support the claim that the animals are germ-free.…”

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

“…In this case, cell vitality is not exclusively related to reproducibility or cultivability and in certain aspects the cell can be described as being in a viable-but-non-culturable state (VBNC) as reviewed by Zhao, Zhong, Wei, Lin, and Ding (2017). A truly "dead" cell may therefore lack the minimal structural integrity and ability to carry out basic cell functionality such as control and activity of physiology, metabolism and genetic material and possess neither vitality or viability characteristics (Davis, 2014).…”

“…Based on these various states, enumeration of bacterial cells has evolved into two general methodologies; (1) culture dependent and (2) culture independent (Davis, 2014). In the former technique, a liquid sample is plated onto solid media, incubated under defined conditions of time and temperature with data expressed as numbers of colony forming units per gram of original material (cfu/g) (Davey, 2011).…”

“…The main advantages of FCM for microbiology are: rapid assay times and data generation (1-2 min), high numbers of cells that can be analysed per sample (10,000 and upwards), minimal sample volume (from 5 μl), potential high throughput, multiplicity of stains available to examine various aspects of cell viability, structure and/or metabolism (multiparametric), and less labour and space required compared with conventional plating techniques (Wilkinson, 2016). Flow cytometry is used within microbiology in an expanding range of applications including; medical diagnostics (Aebisher, Bartusik, & Tabarkiewicz, 2017;Diaz, Herrero, Garcia, & Quiros, 2010), water treatment and supply quality monitoring (De Roy, Clement, Thas, Wang, & Boon, 2012;Egli & Kotzsch, 2015;Hammes et al, 2008), brewing fermentation monitoring (Achilles, Harms, & Müller, 2006;Bühligen et al, 2014;Overton, 2015), pharmaceutical, environmental testing (Herrero & Diaz, 2015), the dairy industry (Doolan, Nongonierma, Kilcawley, & Wilkinson, 2014;Pane, 2013;Sheehan, O'Loughlin, O'Cuinn, FitzGerald, & Wilkinson, 2005;Yanachkina, McCarthy, Guinee, & Wilkinson, 2016) and for manufacturers of probiotic cultures and products (Davis, 2014;Raymond & Champagne, 2015). The general industrial applications of FCM include rapid detection of pathogens, estimation of cell damage following processing steps such as heating, spray drying and freeze drying, and monitoring of bioprocesses (Diaz et al, 2010;Herrero & Diaz, 2015).…”