Quantifying the number of live (dormant or metabolically active) and dead bacteria is critical for assessing the response of a microbial biofilm to surfaces. Current approaches to characterize biofilm viability suffer from count inaccuracy, toxicity, insensitivity to dormant bacteria, and probe concentration dependency. Our objective is to develop a non-destructive imaging method to accurately identify and quantify individual dead and live bacteria in a population. Here we use a slow-response potential-sensitive fluorophore that enters bacteria upon membrane depolarization and binds non-specifically to DNA and proteins, resulting in an increase in fluorescence intensity. However, viability assignment in biofilms based on fluorescence intensity is not reliable due to photobleaching and difficulty in assigning significance to an intensity value with confidence. Fluorescence lifetime is generally independent of concentration, very weakly affected by photobleaching and very sensitive to the fluorophore microenvironment. Fluorescence lifetime imaging (FLIM) of a membrane voltage probe to discriminate “dead” and “dormant” bacteria was demonstrated in a model planktonic system. “Dormant” S. mutans was incubated overnight to stationary phase, washed and resuspended in saline. “Dead” samples were generated by fixation and permeabilization of “dormant” bacteria. “Dead” and “dormant” samples were incubated in the membrane voltage probe Bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)), washed, and resuspended in saline. FLIM results shown below demonstrate distinct separation between dead and dormant lifetime population distributions, indicating this is a promising approach for distinguishing dead bacteria from those that are metabolically dormant. Future work will focus on validating these findings using other relevant planktonic systems and then moving to biofilms.

Fluorescence lifetime imaging of membrane potential probes for distinguishing microbial phenotypes

254th National Meeting (2017)

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