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 milliliter round-bottom tubes containing five milliliters of tryptone soya broth, or TSB, medium, in a 37 degrees Celsius incubator with shaking overnight. When the cultures have reached stationary phase, centrifuge the bacterial cells and re-suspend the pellets and PBS to a 2 times 10 to the ninth colony forming unit per milliliter concentration. Dilute these bacterial suspensions at a 1 to 200 ratio and TSB medium supplemented with zero point five percent glucose and seed 200 microliters of bacterial cells per well into three wells per strain per experimental group in a polystyrene cell-culture treated 96 well microplate for a 24 hour incubation at 37 degrees Celsius, with oxygen, without shaking.

The next day, gently wash the wells three times with PBS without disturbing the biofilms. Next, add 200 microliters of freshly prepared ALA to the appropriate experimental wells, and place the plate at 25 degrees Celsius for one hour protected from light. Then irradiate the plate with a light emitting diode at a 100 milliwatt per square centimeter light intensity for one hour.