The microtiter assay is one of the most widely used methods for assessing biofilm formation. Though it has high throughput, this assay is known for its substantial deviation from experiment to experiment, and even from well to well. Since the assay constitutes one of the pillars of biofilm research, it was decided to examine the wells of a microtiter plate directly during growth, treatment, and the steps involved in crystal violet (CV) measurements.

An inverted Zeiss LSM 880 confocal laser scanning microscope was used to visualize and quantify biomass directly in the wells of the microtiter plate. Green fluorescent protein-tagged Pseudomonas aeruginosa, PAO1, and live/dead stains were used to assess the structure, state, and position of biomass build-up. Microscopic observations were compared with colony-forming unit (CFU) and CV measurements.

The development and the structured architecture of biomass was observed in real-time in the wells. Three-dimensional images of biomass were obtained from all of the microtiter wells; these showed variations from well to well. CV staining showed large variations in remaining biomass, depending on the method selected to remove the supernatant prior to CV staining (i.e. pipetting or manually discarding the fluid by inversion, washed or unwashed wells). Colony-forming unit counts or live/dead staining used to evaluate biomass with or without antibiotic treatment proved imprecise due to aggregation, limited removal of biomass, and overestimation of dead staining.

The highly structured microenvironment of biomass in microtiter wells needs to be considered when designing and analyzing experiments. When using microtiter plates, stochastic variation due to growth and handling may lead to flawed conclusions. It is therefore recommended that this assay be used as a screening tool rather than as a stand-alone experimental tool.