Abstract
A rapid and easy method that takes advantage of an inexpensive and portable fibre-based spectroscopic system (optrode) to determine the ratio of live to dead bacteria is proposed. Mixtures of live and dead Escherichia coli with proportions of live:dead cells varying from 0 to 100% were stained using SYTO 9 and propidium iodide (PI) and measured using the optrode. We demonstrated several approaches to obtaining the proportions of live:dead E. coli in a mixture of both live and dead, from analyses of the fluorescence spectra collected by the optrode. To find a suitable technique for predicting the percentage of live bacteria in a sample, four analysis methods were assessed and compared: SYTO 9:PI fluorescence intensity ratio, an adjusted fluorescence intensity ratio, single-spectrum support vector regression (SVR) and multi-spectra SVR. Of the four analysis methods, multi-spectra SVR obtained the most reliable results and was able to predict the percentage of live bacteria in 108 bacteria/mL samples between c. 7 and 100% live, and in 107 bacteria/mL samples between c. 7 and 73% live. By demonstrating the use of multi-spectra SVR and the optrode to monitor E. coli viability, we raise points of consideration for spectroscopic analysis of SYTO 9 and PI and aim to lay the foundation for future work that uses similar methods for different bacterial species.
Highlights
Monitoring bacterial viability is an important task in many fields of microbiological study including the monitoring of food safety and public health
A count of the viable bacteria in the sample is obtained via enumeration of the colony-forming units (CFU) following an incubation period, with the assumption that each CFU grew from one bacterium of the sample [1]
Grid search was applied to search over various parameter values of both ε and C to find the estimators that minimised the mean squared error of the predictions. This process was optimised by group K-fold cross-validation (GKCV), where the spectral training dataset was split into groups according to the M experiments performed to collect the data at each bacterial concentration, which were 3 and 4 for the 107 and 108 bacteria/mL samples, respectively
Summary
Monitoring bacterial viability is an important task in many fields of microbiological study including the monitoring of food safety and public health. Current standard assessments rely largely on the agar plate count method. Due to the need for incubation, the agar plate count process requires 1 to 5 days [1, 2]. Only the cells that can form colonies under the conditions of the experiment will be counted, . The signals from the dyes are typically measured using fluorescence microscopy, fluorescence-based microplate readers, or flow cytometry. Fluorescence microscopy provides direct morphological information of individual cells, but it has a small field of view; the analysis of large sample volumes becomes time consuming [5, 6]. Fluorescence-based microplate readers and flow cytometry (FCM) have superior ease of use, as many of its operations can be automated and performed in parallel [7, 8]. The equipment is bulky and often requires operation by trained technicians
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