Pipette-based Biofilm Disruption and Quantification

The treatment of established biofilm with NEBB and the resulting disruption of biofilm included the release of planktonic forms into the culture supernatant and the detachment of clumps of bacteria living in biofilm. In order to provide conclusive data on the effects on biofilms, following published methodology [37 (link)], planktonic forms had to be removed from the cultures before staining for biofilm mass and metabolic activity. The removal of planktonic forms and the addition of washing buffer were performed using a very low speed to avoid mechanical removal of biofilm material. The removal of planktonic forms was performed using electronic 12-channel pipettes (Viaflo, Integra, Hudson, NH, USA), where the speed was set to “1” (the maximum speed is “10”). Phosphate-buffered saline was added, also using speed “1”, where the liquid was dispensed onto the sidewalls of each well to avoid disruption of biofilm by direct pipetting actions onto biofilm. For the cultures of Pseudomonas aeruginosa, this pipetting allowed the scoring of slime formation, where “0” indicated no change in viscosity of the culture medium, and a score of “3” (300%) indicated that the entire culture medium had turned into a mucus plug.