LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

My research is focused on exploring different molecules from the family of flavylium compounds both natural and synthetic with a core interest in studying their bioactivity in the context of UV photoprotection and evaluating their potential as photosensitizers for photodynamic therapy applications. The recent findings in our research group demonstrated the light activated properties of amino based flavylium dyes, establishing them as a promising new class of photosensitizers. These compounds exhibit significant potential for further exploration, paving the way for innovative applications in the field.

Using the 96-well microplate and light panel system, we can obtain a high throughput screening of photosensitizers in a controlled environment, simplifying the identification and comparison between different candidates for photodynamic therapy. To begin, introduce the Staphylococcus aureus bacteria on Mueller-Hinton agar plate from the stop culture. Incubate the plate at 37 degrees Celsius for 24 hours to obtain fresh culture.

Collect two to three fresh colonies from the agar plate and dilute them in six milliliters of pre-warmed sterile filtered PBS at pH 7.4. Vortex the inoculum at 1, 200 revolutions per minute for three to four cycles to homogenize the sample and withdraw three milliliters of the homogenized inoculum into a disposable cuvette. After measuring the optical density at 600 nanometers, dilute the sample to match the optical density of 0.1 using PBS.

Prepare a stock solution of the photosensitizer compound in an appropriate solvent such as DMSO or PBS. Dilute the stock solution in PBS to create the working solution, ensuring that the bacterial culture media components do not interfere with light absorption. Vortex the working solution to ensure complete homogenization.

To begin, prepare the Staphylococcus aureus suspension and the photosensitizer compound working solution. Prepare two separate 96-well plates, one designated for light exposure and another for dark control. In each plate, dispense 100 microliters each of the photosensitizer solution and the bacterial suspension.

Mix the solutions 5 to 10 times with the pipette. Incubate the plates at 37 degrees Celsius for 30 minutes to let the photosensitizer interact with the bacterial cells. After incubation, remove the plates from the incubator.

Keep one of the plates protected from light exposure as the dark control. Place the other 96-well plate above the rectangular 50 watt LED panel. Mark the edges of the plate on the LED surface to ensure consistent placement across protocol repetitions.

Expose the plate to LED light for about 15 minutes. Use another 96-well plate to prepare serial dilutions of the samples, including controls, non-irradiated and irradiated wells. Add 180 microliters of PBS to each well, according to the number of samples.

Aliquot 20 microliters from each well in the irradiated and non-irradiated plates into the first column of the PBS-filled plate. Using a multi-channel micro pipette, perform a simple serial dilution from well 1 to 6. Divide an agar plate into six equal sections with each section corresponding to one of the dilutions.

Aliquot 10 microliters from each well onto the corresponding section of the agar plate. Incubate the plate for 18 to 20 hours. Remove the agar plate from the incubator and identify areas suitable for colony counting.

Within each section of the auger plate, count the number of colony forming units. The visible light range spectrum showed three distinct peaks between 400 and 700 nanometers. Under dark conditions, none of the tested flavylium compounds showed cytotoxic effects.

In contrast, after light exposure, the tested compounds caused a dose dependent decrease in Staphylococcus aureus viability with significant effects observed at 6 and 12 micromolar concentrations.