Utilizing glycoside hydrolases to improve the quantitation and visualization of biofilm bacteria

The complexity of microbial biofilms offers several challenges to the use of traditional means of microbial research. In particular, it can be difficult to calculate accurate numbers of biofilm bacteria, because even after thorough homogenization or sonication, small pieces of the biofilm remain, which contain numerous bacterial cells and result in inaccurately low colony forming units (CFU). In addition, imaging of infected tissue ex vivo often results in a disparity between the CFU and the number of bacterial cells observed under the microscope. We hypothesized that this phenomenon is due to the biofilm extracellular polymeric substance decreasing the accessibility of stains and antibodies to the embedded bacterial cells. In this study, we describe incorporating EPS-degrading glycoside hydrolases for CFU determination to obtain a more accurate estimation of the viable cells and for immunohistochemistry to disrupt the biofilm matrix and increase primary antibody binding to the bacterial cells.