A rapid and easy method for determining the ratio of live to dead bacteria in a sample is valuable in many fields of microbiological and pharmacodynamics studies, and for the monitoring of food safety and public health. Efficient, culture-independent detection of live and dead bacteria can be achieved using differentially staining fluorescent dyes SYTO 9 and propidium iodide (PI). Fluorescence microscopy and flow cytometry have been used extensively for detection of these live/dead cell fluorescence signals, however, both these methods require bulky equipment and are relatively expensive to implement. We are developing a cost-effective and portable fibre-based fluorometer for the accurate measurement of fluorescence signals to determine the proportions of live and dead bacteria present. Escherichia coli suspensions with differing ratios (from 0 to 100%) of live to dead cells were stained with SYTO 9 and PI. Fluorescence spectra were collected from samples using the optrode, which consists of a 473 nm solid state laser, DAQ controlled shutter, photodiode for monitoring the laser power and CCD spectrometer. Three regression techniques (simple SYTO 9/PI intensity ratio, adjusted SYTO 9/PI intensity ratio, and support vector regression (SVR)) were used to analyse the spectra to predict the percentage of live bacteria present in each sample. To date, SVR achieved the best correlation (R^2>0.99, for samples containing 108 bacteria/mL). Preliminary results are promising and show that this method may be extended to a wider concentration range or modified for the calibration of other bacterial species of interest.

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Fang Ou, Cushla McGoverin, Simon Swift, and Frédérique Vanholsbeeck, "Rapid monitoring of live to dead bacterial cell ratios using the optrode (Conference Presentation)," Proc. SPIE 10881, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII, 108810Z (Presented at SPIE BiOS: February 06, 2019; Published: 4 March 2019); https://doi.org/10.1117/12.2506213.6008553325001.