Detecting and characterising bacteria as well as monitoring their viability are routine tasks in microbiology. Conventional methods of cell detection and viability monitoring are often time-consuming and/or expensive. We have developed a near-real time, cost-effective and portable fluorometer, the optrode, for quantifying fluorescence signals. We are currently developing protocols to use the optrode to detect, identify and quantify bacteria as well as to monitor their viability using nucleic acid stains such as SYTO 9 and propidium iodide that are routinely used as live/dead stains. Our results are promising but indicate that better dyes are needed to fully characterise bacteria.
Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We investigated live/dead spectrometry using a fluorimeter (Optrode) as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. Killing was detected by the Optrode in near real-time when E. coli was treated with ampicillin and stained with SYTO 9 and/or PI but only when a suitable concentration, bacterial growth phase, and treatment time was used.
A rapid and easy method for the monitoring of both live and 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 optimising a method to determine live and dead bacterial concentration that takes advantage of an inexpensive fibre-based fluorimeter, the optrode, which can measure fluorescence intensity in bacterial solutions in challenging working environments. The concentrations of live and dead bacteria were predicted using multivariate analysis of optrode-measured fluorescence spectra, which is then compared with results obtained from the flow cytometry measurements. The findings from this study will be used to establish a general method for the monitoring of live and dead bacteria using the optrode.
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