Dead/alive bacteria detection using an all-fibre optical system.
2014
Accurate monitoring of microbial viability plays an essential role in pharmacodynamic studies such as in
estimating the efficiency of antimicrobial agents. Traditionally, bacterial viability is determined by their ability
to form colonies on solid growth medium or to proliferate in liquid nutrient broths but, with these culture-based
methods, the live bacterial population can only be estimated retrospectively.
To address this challenge, we have employed differential fluorescence staining and an all-fiber optical system
developed by our group. The detection is based on the collection of the fluorescence from commercial dyes that
produce a substantially increased signal upon binding with bacterial nucleic acids. The dyes allow
discrimination between alive and dead cells through differential membrane permeability and fluorescence
wavelength. The respective fluorescence signal is correlated to the number of bacterial cells present in the
sample.
Our setup uses DPSS lasers and a sensitive CCD-based spectrometer over the 400-800 nm wavelength range. A
laser shutter allows the sample exposure time and acquisition time to be synchronized to minimize the effect of
photobleaching.
As a model, bacteria (Escherichia coli or Staphylococcus aureus) killed with isopropyl alcohol were mixed with
live cells at different ratios. The population ratios of alive and dead cells were accurately quantified by our
optical setup providing a rapid method for the estimation of bactericidal treatments.
In summary, our optical system may offer a robust, accurate and fast alternative for detection of dead/alive
bacteria in turbid solution opening the new avenues for pharmacodynamic studies.
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