Citric acid (Sigma-Aldrich) at a concentration of 10 mg/mL prepared using LB medium was added to each droplet in varying volumes via a hydrophilically modified glass capillary connected to a syringe pump so that the final concentration in each droplet ranging from 0 to 9 mg/mL propidium iodide (PI) (Sigma-Aldrich) was added to Citric acid to a final concentration of 60 µM to visualize the dead bacteria in biofilms. The Citric acid–PI mixture was added to the microarrays and incubated for 2 h to allow for the action of Citric acid on the bacteria and staining of the dead cells by PI.
The droplets were then imaged using a FluoView 1000 confocal microscope (Olympus) with a 4× objective. Three image channels, GFP 488, Alexa 594, and bright field, were acquired from each droplet. The 3D image stacks of the representative droplets were also acquired to visualize the biofilm structure within the droplets before and after treatment with the Citric acid.
The droplets were then imaged using a FluoView 1000 confocal microscope (Olympus) with a 4× objective. Three image channels, GFP 488, Alexa 594, and bright field, were acquired from each droplet. The 3D image stacks of the representative droplets were also acquired to visualize the biofilm structure within the droplets before and after treatment with the Citric acid.