Precise quantification of viable probiotic cells is critical for dose-response relationships in gastrointestinal physiology, where microbial viability directly influences colonization potential and subsequent health outcomes

Novel strategies to improve the accuracy of bacterial enumeration remain a topic of interest in microbiological research. Accurate quantification of viable probiotics is essential for linking microbial dosage to physiological and metabolic effects in the gastrointestinal tract, informing both clinical efficacy and product labeling for desired health outcomes. While traditional nutrient agar plate count enumeration (plate count) methods are frequently implemented for determining colony-forming units (CFUs), molecular techniques such as droplet digital polymerase chain reaction (ddPCR) are emerging as promising, more targeted alternatives. It is understood that the equivalence of ddPCR to CFU-based enumerations can vary and remains under investigation. This study evaluated the agreement between plate count and ddPCR for quantifying viable probiotics in a complex nutritional supplement matrix. A probiotic mixture of 5 unique strains were added to the nutritional supplement AG1, consisting of prebiotics, phytonutrients, and a multivitamin-mineral complex, to create a representative product matrix for analysis. The blend was tested at five different probiotic dose levels: 4 mg, 37 mg, 278 mg, 648 mg, and 1000 mg corresponding to 1B, 10B, 75B, 175B, and 270B CFU, respectively. All tests were run in triplicate. A standard plate count technique, with minor modifications, was employed to measure CFU utilizing a nutrient rich MRS agar via the pour plate method. Plates were incubated at 38° C under anaerobic conditions for 48 hours, and colonies were manually counted. In parallel, ddPCR was employed to measure live cell counts. Prior to PCR amplification, samples underwent filtration and pelleting steps to remove excess debris from the samples, and a combination of propidium monoazide (PMA) and ethidium monoazide (EMA) was used to selectively exclude DNA present in the samples from non-viable cells. PMA and EMA exclude DNA from PCR amplification based on membrane integrity and metabolic potential respectively. Linear regression analysis was employed to evaluate the agreement between enumeration methods against expected values across all dose levels and paired t-tests were used to compare values between each method. Results demonstrated no statistical difference (p < 0.05) observed between plate count and ddPCR-based live cell counts at each dose tested. Linear regression analysis revealed high goodness of fit between actual cell counts and expected counts for pate enumeration (R2 = 0.9547) and ddPCR (R2 = 0.9712). Together, these data indicate that ddPCR provides a statistically equivalent, and potentially more precise, method for quantifying viable fresh probiotics in complex supplement matrices. The slightly higher coefficient of determination for ddPCR supports its use as a reliable, valid, and potentially more precise enumeration method, particularly in quality control applications where accurate probiotic dosing is critical for ensuring physiological efficacy and clinical relevance. Nevertheless, further research is warranted to evaluate the comparative performance of these methods across the natural shelf life of nutritional supplement products. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.