Abstract:
Biophotonics is a multidisciplinary research area encompassing application of light-based technologies to the life sciences and medicine. In the realm of infectious disease research, the majority of the work has focused on utilisation of fluorescent dyes to detect and quantify viability and other physiological states with measurements taken using a fluorometer, flow cytometer, or fluorescent microscope. The available viability fluorescent dyes inform on specific cell processes that reflect a basic aspect of bacterial cell life, such as an intact cell membrane as indicated by membrane integrity. However, application of these dyes to bacteria exposed to a wide range of inimical processes has demonstrated that they are not always adequate for live/dead determination. Investigation of bacterial physiology using absorbance is an underexploited biophotonics approach due to the limitations imposed by the associated scattering of light from turbid bacterial samples. We have employed a novel, next-generation spectroscopy instrument called the CloudSpec, developed at the Victoria University of Wellington, which permits turbid media to be analysed quickly and accurately. The CloudSpec eliminates the influence of scattering allowing for a direct measurement of absorbance and generation of an absorbance spectrum. We have explored the potential to use CloudSpec derived absorbance spectra to detect near real-time loss of Escherichia coli viability accrued from lethal challenges, which include 70 % isopropanol and the antibiotics, ampicillin and ciprofloxacin. We have also investigated the influence of staining with fluorescent membrane integrity dyes allowing for comparison with measurements taken with a fluorometer developed at The University of Auckland called the Optrode. Preliminary results suggest that there are spectral differences between the absorbance of lethally challenged cells and viable cells for both unstained and stained cell samples. The collected spectra also gave insight into dye excitation and emission characteristics, which complement and supplement corresponding fluorescence measurements taken with the Optrode.