(596aa) Confocal Fluorescent Microscopy Vs. Direct Enumeration for Quantification of Biofilm Infection Deactivation

Development of strategies to mitigate bacterial biofilm infections is hampered by the difficulty in observing and quantifying biofilm viability.  We are investigating thermal deactivation of Pseudomonas aeruginosa biofilms at a variety of time and temperature combinations.  Eradication has been evaluated both through confocal fluorescent microscopy using membrane permeability dyes and through direct enumeration of colony forming units cultured from sonically homogenized post-treatment biofilms.  Microscopy, while providing much more rapid, direct feedback, is limited by the ambiguity of intermediate light intensities, where it is unclear whether the light is due to fluorescing biomass in the confocal plane, or whether the light is simply background from extracellular or out-of-plane material.  This problem is particularly acute at low signal levels, i.e. when only tiny but important fractions of viable bacteria remain.  This presentation will discuss the applicability of image processor-based quantification of bacterial biofilms using both subjective and objective light thresholding, as compared to direct enumeration.