Light-Activated Treatment of Bacterial Biofilms Using a Photosensitizer Precursor

Begin with microplate wells containing biofilms of a pathogenic bacterium, divided into four experimental groups.

Gently aspirate the medium and rinse with a buffer to remove non-adherent cells.

Add a buffer containing a photosensitizer precursor to two groups, while the remaining two receive buffer alone.

Incubate all groups in the dark to allow the precursor to diffuse through the biofilm, enter the bacterial cells, and get metabolized into a photosensitizer that accumulates within the cytoplasm and the membranes.

After incubation, expose one precursor-treated group and one buffer-only group to light irradiation.

In the precursor-treated group, the light excites the photosensitizer, which transfers energy to molecular oxygen and generates reactive oxygen species (ROS).

The ROS disrupt membranes, degrade proteins, and fragment DNA, resulting in bacterial cell death.

The other three conditions fail to produce ROS, as either the precursor or light is absent, leaving the biofilm unaffected.

To generate a biofilm in a 96 well plate, first inoculate one minus 80 degrees Celsius thawed Staphylococcus aureus USA three hundred in three biofilm forming clinical strain cultures in individual 14 mil