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IntroductionProbiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. From this definition, accurate enumeration of probiotic products is a necessity. Nonetheless, this definition does not specify the methods for assessing such viability. Colony forming units is the de facto gold standard for enumerating viable in probiotic products. The notion of microbial viability has been anchored in the concept of cultivability, which refers to a cell’s capacity to replicate and form colonies on agar media. However, there is a growing consensus that the term “viability” should not be exclusively tied to the ability to cultivate cells. For example, bacterial cells can exist in a Viable But Non-Culturable (VBNC) state, characterized by the maintenance of characteristics such as membrane integrity, enzymatic activity, pH gradients, and elevated levels of rRNA, despite losing the ability to form colonies.MethodsHerein we present the results of a collaborative inter-laboratory ring test for cytometric bacterial quantification. Specifically, membrane integrity fluorescence flow cytometry (FFC) method and the newer impedance flow cytometry (IFC) method have been used. Both methods interrogate single cells in solution for the presence of intact membranes. FFC exploits fluorochromes that reflect the presence or absence of an intact membrane. IFC probes membrane integrity in a label-free approach by detecting membrane-induced hindrances to the propagation of electricity.ResultsA performance ring-test and comparison design on the FFC method showed that the method is robust against the exchange of equipment, procedures, materials, and operators. After initial method optimization with assessments of rehydration medium, wake-up duration, and phase shift gating on the individual strains, the IFC method showed good agreement with the FFC results. Specifically, we tested 6 distinct species of probiotic bacteria (3 Lactobacillus and 3 Bifidobacterium strains) finding good agreement between FFC and IFC results in terms of total and live cells.DiscussionTogether, these results demonstrate that flow cytometry is a reliable, precise, and user-friendly culture-independent method for bacterial enumeration.

Recent acquisitions about the role of the microbiota in the functioning of the human body make it possible to envisage an increasing use of beneficial microbes, and more particularly of probiotics as well as their metabolites, as nutritional supplements. National and EU authorities are engaged in assuring the safety and quality of food supplements and in defining rules to assess and communicate their efficacy on human health. The quality of probiotics, intended as strains’ identification, viability, and stability over time, is a crucial factor of credibility with consumers and health professionals. Analytical technologies for the quality control of probiotics must also be adapted to new preparations, such as those including new multistrains complex combinations. Accredited laboratories face this relevant challenge on a daily basis. Through its close collaboration with the laboratory commissioned to produce the specifications for its ESLP quality label (identification and quantitative analyses) together with its scientific committee, the ESLP has been focusing on this issue for 10 years. Recently, as part of the internationalization of the ESLP quality label, a new and unique initiative in Europe for the evaluation of the quality of probiotic preparations has been carried out. The collaboration between two accredited laboratories in Belgium and in Italy represented a concrete example of supranational collaboration in the assessment of the quality of probiotic preparations. Results show that both laboratories are in line as expected in terms of performance. Common approaches to the qualitative assessment of probiotic preparations, especially for complex and composite recipes, in terms of number of strains and included substances, should be encouraged and promoted all over the EU.