Accurate characterization of submicron particles within biological fluids presents a major challenge for a wide range of biomedical research. Detection, characterization and classification are difficult due to the presence of particles and debris ranging from single molecules up to particles slightly smaller than cells. Especial interest arises from extracellular vesicles (EVs) which are known to play a pivotal role in cell-signaling in multicellular organisms. Tunable resistive pulse sensing (TRPS) is increasingly proving to be a useful tool for high throughput particle-by-particle analysis of EVs and other submicron particles. This study examines the capability of TRPS for characterization of EVs derived from bacteria, also called outer membrane vesicles (OMVs). Measurement of a size distribution (124 ± 3 nm modal diameter) and concentration (lower bound 7.4 x 109 mL-1) are demonstrated using OMVs derived from uropathogenic Escherichia coli. Important aspects of measurement are discussed, including sample preparation and size selection. Application of TRPS to study EVs could assist the development of these particles in clinical diagnostics and therapeutics.
KEYWORDS: Luminescence, Bacteria, Spectroscopy, Data acquisition, Camera shutters, Signal to noise ratio, Diode pumped solid state lasers, Fiber couplers, Ocean optics, Charge-coupled devices
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.
KEYWORDS: Luminescence, Bacteria, Spectroscopy, Microorganisms, Fluorescence spectroscopy, Data acquisition, Camera shutters, Signal to noise ratio, Principal component analysis, Charge-coupled devices
Enumeration of microorganisms is an essential microbiological task for many industrial sectors and research fields. Various tests for detection and counting of microorganisms are used today. However most of the current methods to enumerate bacteria require either long incubation time for limited accuracy, or use complicated protocols along with bulky equipment. We have developed an accurate, all-fibre spectroscopic system to measure fluorescence signal in-situ. In this paper, we examine the potential of this setup for near real time bacteria enumeration in aquatic environment. The concept is based on a well-known phenomenon that the fluorescence quantum yields of some nucleic acid stains significantly increase upon binding with nucleic acids of microorganisms. In addition we have used GFP labeled organisms. The fluorescence signal increase can be correlated to the amount of nucleic acid present in the sample. In addition we have used GFP labeled organisms. Our results show that we are able to detect a wide range of bacteria concentrations without dilution or filtration (1-108 CFU/ml) using different optical probes we designed. This high sensitivity is due to efficient light delivery with an appropriate collection volume and in situ fluorescence detection as well as the use of a sensitive CCD spectrometer. By monitoring the laser power, we can account for laser fluctuations while measuring the fluorescence signal which improves as well the system accuracy. A synchronized laser shutter allows us to achieve a high SNR with minimal integration time, thereby reducing the photobleaching effect. In summary, we conclude that our optical setup may offer a robust method for near real time bacterial detection in aquatic environment.
We developed a computerized optical probe for near real time bacterial detection in water. This microorganism detection technique, based on fluorescence enhanced by nucleic acids staining, shows promising results compared to conventional methods.
We investigate different fibre probes for fluorescence measurements. Our design shows a threefold sensitivity
improvement compared to a single fibre probe allowing bacteria concentration as low as 1 CFU/ml to be measured.
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