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

TL;DR

This paper presents a rapid, cost-effective fibre-based spectroscopic method for on-site enumeration of live and dead bacteria using fluorescent dyes and spectral analysis techniques, suitable for real-time microbial monitoring.

Contribution

The study introduces a novel optrode-based spectroscopic device combined with PCR analysis for accurate, rapid bacterial enumeration without sample pre-concentration.

Findings

PCR reliably quantified bacteria from 10^8 to 10^6.2 bacteria/mL.

Detection limit for live bacteria was as low as 10^5.7 bacteria/mL.

The method effectively distinguishes live and dead bacteria populations.

Abstract

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.