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Isothermal microcalorimetry (IMC) is a potent analytical method for the real-time assessment of microbial metabolic activity, which serves as an indicator of microbial viability. This approach is highly relevant to the fields of probiotics and Live Biotherapeutic Products (LBPs), offering insights into microbial viability and growth kinetics. One important characteristic of IMC is its ability to measure microbial metabolic activity separately from cellular enumeration. This is particularly useful in situations where continuous tracking of bacterial activity is challenging. The focus on metabolic activity significantly benefits both probiotic research and industrial microbiology applications. IMC’s versatility in handling different media matrices allows for the implementation of viability assessments under conditions that mirror those found in various industrial environments or biological models. In our study, we provide a proof of concept for the application of IMC in determining viability and growth dynamics and their correlation with bacterial count in probiotic organisms. Our findings reinforce the potential of IMC as a key method for process enhancement and accurate strain characterization within the probiotic sector. This supports the broader objective of refining the systematic approach and methods used during the development process, thereby providing detailed insights into probiotics and LBPs.

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.