FAST ATTACHMENT QUANTIFICATION: A NEW METHOD FOR REAL TIME HIGH THROUGHPUT QUANTIFICATION OF BACTERIAL ATTACHMENT

Bacterial attachment is a subject of interest, but to-date no satisfactory method is available for its kinetic measurement is currently available. Microscopy is labor intensive and the small observation filed makes for poor statistics; microtiter based methods like crystal violet and XTT are destructive and have poor time resolution; QCM-D (quartz crystal microbalance with dissipation) is indirect and requires specialized equipment and expertise. We have developed a novel method that enable fast quantification of attachment kinetics based on GFP expressing bacteria, and on masking the fluorescence of unattached bacteria by addition of a suitable dye, thus limiting fluorescence to the layer just above the bottom of a well. Using a standard plate fluorometer we were able to obtain parallel kinetic measurement of bacterial attachment in time resolutions of seconds and minutes. Results are in full agreement with established methods, while the real-time kinetics data offers some interesting questions regarding the mechanism by which clinically used antibiotics effect on biofilm formation.