Hanan R Shehata ^1* Marco Pane ² Elna Buys ³ Binu Koshy ⁴ Christina Vegge ⁵ Jean L Schoeni ^6*

• ¹ Purity-IQ Inc, Guelph, Ontario, Canada
• ² Probiotical Research srl, Novara, Italy
• ³ Department of Consumer and Food Science, University of Pretoria, Hatfield, South Africa
• ⁴ Dietary Supplements and Herbal Medicines, United States Pharmacopeia, Rockville, MD, United States
• ⁵ Probi AB, Lund, Sweden
• ⁶ Eurofins Microbiology Laboratory, Madison, WI, United States

The live microorganism industry is rapidly growing, producing probiotics and live biotherapeutic products (LBPs) designed to deliver health benefits. Ensuring these products contain viable, strain-specific microorganisms at effective levels is essential, but accurately measuring viability and potency remains challenging.Colony-forming unit (CFU) enumeration, the traditional gold standard, relies on a cell's ability to form colonies on culture media. While widely used, it has significant limitations. CFU methods fail to account for viable but non-culturable (VBNC) cells, which maintain metabolic activity but cannot grow on culture media. Moreover, CFU enumeration often falls short for probiotic blends, as strains with varying growth requirements or interactions may not form colonies under standardized conditions.With growing consumer awareness and stricter regulatory demands, more accurate and comprehensive enumeration techniques are needed. Emerging methods such as flow cytometry, real-time PCR, digital PCR and advanced imaging assess viability based on cellular activity rather than replication alone. These approaches offer reliable assessments of complex probiotic formulations, ensuring higher product quality and efficacy.Adopting advanced techniques is critical to meet regulatory standards, enhance product reliability, and build consumer trust, marking a significant step forward in ensuring the health benefits of live microorganism products. longum subsp. longum UABl-14. High specificity, reaction efficiency, and precision were demonstrated. The method enabled stability monitoring of the target strain in multi-strain finished products during storage, which cannot be achieved using plate count methods. The FCC ring test demonstrated robustness across changes in equipment, procedures, materials, and operators. After a one-time per strain optimization, the IFC method showed good agreement with FCC results. Combined, the ring test and comparison results indicated that these culture-independent flow cytometry methods saved time, were reliable, precise, adaptable to bacterial enumeration, and allowed exploration of viability. ICM captures changes in heat produced by living organisms (e.g., metabolic processes).Morazzoni et. al. contributed a proof-of-concept study featuring the application of IMC to determine viability and growth dynamics and its correlation to the plate counts for Lacticaseibacillus rhamnosus and Limosilactobacillus fermentum. Experiments established suitability of ICM for viability assessment and enumeration of probiotic products. Relationships between ICM and plate counting were determined via standard curves and linear regression analyses. Method robustness was observed through the maintenance of correlations between time-to-peak (TTP) heat detected in ICM and CFU/mL from plate counting across various culture conditions. Finally, IMC, flow cytometry, and acidification measurement experiments were conducted under diverse conditions to demonstrate how IMC can be used as a complementary approach that extends understanding of microbial activity and viability. In 2021, Yang et al. introduced the application of tetrazolium-based colorimetric cell counting kits (CCK-8) to live bacteria. Health and clinical scientists have adopted CCK-8 to enumerate viable probiotics (Chang et al., 2024, Sudan et al., 2022, Xu et al., 2023, Yue et al., 2022). Here,