Microtiter plates are one of the most widely used platforms for biofilm studies. However, these systems have been used rather blindly without knowledge of the flow characteristics inside them. Plates with 96-wells were used to study flow dynamics and its influence on Escherichia coli JM109(DE3) biofilm formation. Two different glucose concentrations (1 g L-1 and 0.25 g L-1) and two different shaking conditions were tested using incubators with orbital diameters of 50 and 25 mm. Biofilm growth was monitored for 60 h and the hydrodynamics were simulated by computational fluid dynamics. These simulations have shown that wall shear stress was higher near the air/liquid interface and average values of 0.070 and 0.034 Pa were obtained for the largest and smallest orbital diameter. For the high glucose concentration, the maximum biofilm amount was attained at 24 h and similar values were obtained in both incubators. For the low glucose concentration, lower values were attained. Numerical simulations indicated that microtiter plates can adequately model biofilm formation in relevant biomedical systems. (c) 2013 Elsevier Ltd. All rights reserved.