Dynamic monitoring of Staphylococcus epidermidis biofilm formation using a microfluidic approach

The crystal violet (CV) assay was the standard for quantifying biofilm biomass but is limited by its inability to replicate dynamic environmental conditions. In contrast, microfluidic flow cells provided a promising alternative, enabling real-time monitoring of biofilm development under controlled and continuously changing environments. This study evaluated the performance of different methods for assessing biofilm formation under various growth conditions, using Staphylococcus epidermidis as a model bacterium. S. epidermidis biofilms are particularly challenging in biomaterial-related infections, highlighting the need for accurate methods to assess biofilm dynamics in such contexts. Biofilm formation was evaluated using the CV assay (adapted from ISO 4768) and single-use polydimethylsiloxane (PDMS) microfluidic flow cells. These flow cells featured dual identical channels, which were individually operated by syringe pumps. In the microfluidic system, inoculum of varying concentrations and types were tested with a standard culture medium. This medium was either supplemented with 0.5% glucose or diluted 1/5 to assess its effects on biofilm development. The CV assays showed similar biofilm growth in both standard and glucose-supplemented media. However, the microfluidic system revealed that glucose was detrimental to biofilm formation. The diluted medium condition impacted biofilm formation significantly with the CV assay but had minimal effect in microfluidics. Biofilm-like structures were consistently detected using microfluidics, even under diluted or low-inoculum conditions, whereas the CV assay failed to observe them. The microfluidic approach enabled real-time biofilm monitoring, offering greater sensitivity to medium composition and inoculum effects, while the CV assay reflects static conditions where environmental changes are mediated by biofilm itself. This study highlights the importance of adopting a dynamic approach to studying biofilm growth mechanisms. The microfluidic approach presented in this study was developed for blood bank applications to contribute to advancements in knowledge on transfusion safety in hospital settings. • Microfluidics allows the study of biofilm in a dynamic growth environment • Real-time analysis of biofilm-like structure formation is enabled by microfluidics • The imaging method allows isolating the environmental parameters that can influence biofilm growth • Crystal violet assay complements the microfluidics method for comprehensive biofilm analysis