Real-Time Monitoring Of Bacterial Growth and Biofilm Formation With Surface Plasmon Resonance Imaging

In this study, for the first time we used a Surface Plasmon Resonance imaging (SPRi) system to monitor bacteria in real time on the sensor surface as they move, grow and form biofilms. SPRi provides label-free and sensitive detection of changes in refractive index that take place within approximately 200 nm of the gold sensor surface. Changes in the refractive index are caused by biomolecules displacing water at the sensor surface. Difference images are generated over time, which show the changes that occur at the surface during the experiment when compared to a reference image chosen at the beginning of the experiment. Combining microfluidics with our SPRi system provides added control while studying bacterial behavior. By placing microchannels on the surface of the gold surface we limit the bacteria to two dimensions and keep them close to the surface, which allows us to study bacterial growth and movement inside the channels.

Two different strains of Pseudomonas aeruginosa PA14 were used for this experiment: Wild Type, which produce biofilm, and PelA mutant, which grow without producing biofilm. The two bacteria were injected into separate microchannels and placed into the SPRi device. The instrument obtained a difference image of the sensor surface every three seconds. We continuously monitored the channels for up to 24 hours as bacteria grew and formed biofilms. We were able to differentiate between the wild type bacteria and the mutant strain. The wild type bacteria attached to the surface and quickly formed large bright spots as they produced extracellular matrix around them, while the mutant bacteria attached to the surface and grew as small discrete bright spots. For the first time, without functionalizing the surface we continuously monitored two different strains of the same bacteria in a single experiment over 1-cm-square area of the sensor surface. This study provides valuable information about bacterial growth and biofilm formation. This new technique can be used to study the effect of different chemicals and surface chemistries on bacterial growth behavior.