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Towards Absolute Viability Measurements for Bacteria



Joy Dunkers, Hariharan K. Iyer, Brynna H. Jones, Charles Camp, Stephan J. Stranick, Nancy Lin


Quantifying viable, vegetative bacteria is a critical measurand in healthcare diagnostics, food safety, and antimicrobial development. Viability determination has traditionally relied on such techniques as plate counting, colorimetric or fluorescent metabolic assays and membrane integrity assays. There are well documented caveats with all of the aforementioned approaches, particularly when differentiating quiescent bacteria from dead. Therefore, we aim to develop a quantitative viability method that can distinguish individual quiescent cells from dead, provide results within hours, and is referenceable to a standard unit of measurement for comparability. In this work, we demonstrate that fluorescence lifetime imaging of an anionic, fluorescent membrane voltage probe fulfills these requirements for a model oral bacteria, Streptococcus mutans. To quantify the results, we developed a random forest machine learning model to classify bacteria into 3 populations: stationary phase (quiescent) (SP), heat killed (HK), and dead via chemical fixation (FP) in saline and phosphate buffered saline. To strengthen our classification, we compared the results to intensity classification using three other models: fluorescence lifetime variables (t1, t2, p1), phasor variables (G, S) or all five variables (t1, t2, p1, G, S). The intensity models had the most objects misclassified and the majority of them were from the SP and FP conditions. The 5 variable model had the most success at classification, with a significant number of SP bacteria predicted to be HK in the saline buffer. Further analysis of the predicted HK bacteria in the SP condition supports the idea that a substantial portion of the population died in culture during prolonged storage prior to imaging. This initial work affirms the potential for using fluorescence lifetime of a membrane voltage probe as a viability marker for quiescent bacteria, and future efforts on other bacterial species
Journal of Biophotonics


viability, fluorescence lifetime microscopy, membrane voltage, bacteria


Dunkers, J. , Iyer, H. , Jones, B. , Camp, C. , Stranick, S. and Lin, N. (2021), Towards Absolute Viability Measurements for Bacteria, Journal of Biophotonics, [online],, (Accessed June 22, 2024)


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Created September 12, 2021, Updated October 28, 2021