arXiv subjects

Simon Swift

Publications and source records attributed to Simon Swift.

3 recordsLinked to original sources

arXiv2019

Rapid and cost-effective evaluation of bacterial viability using fluorescence spectroscopy

The fluorescence spectra of bacterial samples stained with SYTO 9 and propidium iodide (PI) were used to monitor bacterial viability. Stained mixtures of live and dead Escherichia coli with proportions of live:dead cells varying from 0 to 100% were measured using the optrode, a cost effective and convenient fibre-based spectroscopic device. 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 10^8 bacteria/mL samples between c. 7% and 100% live, and in 10^7 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 use similar methods for different bacterial species.

q-bio.QM↗

arXiv2018

Near real-time enumeration of live and dead bacteria using a fibre-based spectroscopic device

A rapid, cost-effective and easy method that allows on-site determination of the concentration of live and dead bacterial cells using a fibre-based spectroscopic device (the optrode system) is proposed and demonstrated. Identification of live and dead bacteria was achieved by using the commercially available dyes SYTO 9 and propidium iodide, and fluorescence spectra were measured by the optrode. Three spectral processing methods were evaluated for their effectiveness in predicting the original bacterial concentration in the samples: principal components regression (PCR), partial least squares regression (PLSR) and support vector regression (SVR). Without any sample pre-concentration, PCR achieved the most reliable results. It was able to quantify live bacteria from $10^{8}$ down to $10^{6.2}$ bacteria/mL and showed the potential to detect as low as $10^{5.7}$ bacteria/mL. Meanwhile, enumeration of dead bacteria using PCR was achieved between $10^{8}$ and $10^{7}$ bacteria/mL. The general procedures described in this article can be applied or modified for the enumeration of bacteria within populations stained with fluorescent dyes. The optrode is a promising device for the enumeration of live and dead bacterial populations particularly where rapid, on-site measurement and analysis is required.

q-bio.QM↗

arXiv2017

Absolute bacterial cell enumeration using flow cytometry

Aim: To evaluate a flow cytometry protocol that uses reference beads for the enumeration of live and dead bacteria present in a mixture. Methods and Results: Mixtures of live and dead Escherichia coli with live:dead concentration ratios varying from 0 to 100% were prepared. These samples were stained using SYTO 9 and propidium iodide and 6 {\mu}m reference beads were added. Bacteria present in live samples were enumerated by agar plate counting. Bacteria present in dead samples were enumerated by agar plate counting before treatment with isopropanol. There is a linear relationship between the presented flow cytometry method and agar plate counts for live (R2 = 0.99) and dead E. coli (R2 = 0.93) concentrations of ca. 104 to 108 bacteria ml-1 within mixtures of live and dead bacteria. Conclusions: Reliable enumeration of live E. coli within a mixture of both live and dead was possible for concentration ratios of above 2.5% live and for the enumeration of dead E. coli the lower limit was ca. 20% dead. Significance and Impact of the Study: The ability to obtain absolute cell concentrations is only available for selected flow cytometers, this study describes a method for accurate enumeration that is applicable to basic flow cytometers without specialised counting features. By demonstrating the application of the method to count E. coli, we raised points of consideration for using this FCM counting method and aim to lay the foundation for future work that uses similar methods for different bacterial strains.

q-bio.QM↗