Given the significant impact of biofilms on human health and material corrosion, research in this field urgently needs more accessible techniques to facilitate the testing of new control agents and general understanding of biofilm biology. Microtiter plates offer a convenient format for standardized evaluations, including high-throughput assays of alternative treatments and molecular modulators. This study introduces a novel Biofilm Analysis Software (BAS) for quantifying biofilms from microtiter plate images. We focused on early biofilm growth stages and compared BAS quantification to common techniques: direct turbidity measurement, intrinsic fluorescence detection linked to pyoverdine production, and standard crystal violet staining which enables image analysis and optical density measurement. We also assessed their sensitivity for detecting subtle growth effects caused by cyclic AMP and gentamicin. Our results show that BAS image analysis is at least as sensitive as the standard method of spectrophotometrically quantifying the crystal violet retained by biofilms. Furthermore, we demonstrated that bacteria adhered after short incubations (from 10 min to 4 h), isolated from planktonic populations by a simple rinse, can be monitored until their growth is detectable by intrinsic fluorescence, BAS analysis, or resolubilized crystal violet. These procedures are widely accessible for many laboratories, including those with limited resources, as they do not require a spectrophotometer or other specialized equipment.

Biofilms can increase pathogenic contamination of drinking water, cause biofilm-related diseases, alter the sediment erosion rate, and degrade contaminants in wastewater. Compared with mature biofilms, biofilms in the early-stage have been shown to be more susceptible to antimicrobials and easier to remove. Mechanistic understanding of physical factors controlling early-stage biofilm growth is critical to predict and control biofilm development, yet such understanding is currently incomplete. Here, we reveal the impacts of hydrodynamic conditions and microscale surface roughness on the development of early-stage Pseudomonas putida biofilm through a combination of microfluidic experiments, numerical simulations, and fluid mechanics theories. We demonstrate that early-stage biofilm growth is suppressed under high flow conditions and that the local velocity for early-stage P. putida biofilms (growth time < 14 h) to develop is about 50 μm/s, which is similar to P. putida\'s swimming speed. We further illustrate that microscale surface roughness promotes the growth of early-stage biofilms by increasing the area of the low-flow region. Furthermore, we show that the critical average shear stress, above which early-stage biofilms cease to form, is 0.9 Pa for rough surfaces, three times as large as the value for flat or smooth surfaces (0.3 Pa). The important control of flow conditions and microscale surface roughness on early-stage biofilm development, characterized in this study, will facilitate future predictions and managements of early-stage P. putida biofilm development on the surfaces of drinking water pipelines, bioreactors, and sediments in aquatic environments.