Dead/alive bacteria detection using an all-fibre optical system

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.