Real-time detection of viable microorganisms by intracellular phototautomerism

BMC Biotechnology

Methodology Article

Alie de Kat Angelino-Bart¹  Frank Schuren¹  Roy Montijn¹  Andreas Nocker¹  Remco Kort¹  Sjaak van Veen²  Herman Verheij³ 

[1] Business Unit Food and Biotechnology Innovations, Microbial Genomics Group, TNO Quality of Life, P.O. Box 360, Utrechtseweg 48, 3700AJ, Zeist, The Netherlands;Business Unit Quality and Safety, Analytical Research Group, TNO Quality of Life, P.O. Box 360, Utrechtseweg 48, 3700AJ, Zeist, The Netherlands;Pyxis Discovery, Delftechpark 26, 2628, XH Delft, The Netherlands;Top Institute Pharma, Galileiweg 8, 2333, BD Leiden, The Netherlands;

关键词: Salicylic Acid;    Fungal Spore;    Sorbic Acid;    Bacterial Spore;    Intracellular Acidification;   

DOI  :  10.1186/1472-6750-10-45

 received in 2009-12-16, accepted in 2010-06-18,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundTo date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner.ResultsThe developed method includes exposure of cells to a weak acid probe at low pH. The neutral probe rapidly permeates the membrane and enters the cytosol. In dead cells no signal is obtained, as the cytosolic pH reflects that of the acidic extracellular environment. In live cells with a neutral internal pH, the probe dissociates into a fluorescent phototautomeric anion. After reaching peak fluorescence, the population of live cells decays. This decay can be followed real-time as cell death coincides with intracellular acidification and return of the probe to its uncharged non-fluorescent state. The rise and decay of the fluorescence signal depends on the probe structure and appears discriminative for bacteria, fungi, and spores. We identified 13 unique probes, which can be applied in the real-time viability method described here. Under the experimental conditions used in a microplate reader, the reported method shows a detection limit of 106 bacteria ml-1, while the frequently used LIVE/DEAD BacLight™ Syto9 and propidium iodide stains show detection down to 106 and 107 bacteria ml-1, respectively.ConclusionsWe present a novel fluorescence-based method for viability assessment, which is applicable to all bacteria and eukaryotic cell types tested so far. The RTV method will have a significant impact in many areas of applied microbiology including research on biocidal activity, improvement of preservation strategies and membrane permeation and stability. The assay allows for high-throughput applications and has great potential for rapid monitoring of microbial content in air, liquids or on surfaces.

【 授权许可】

CC BY   
© Kort et al; licensee BioMed Central Ltd. 2010