This study investigates the mechanism of bacterial inactivation using reactive oxygen species (ROS) generated from L-methionine and flavin mononucleotide (FMN) under violet light irradiation. The method demonstrates a novel approach to reduce bacterial viability through ROS-mediated damage.
Begin with a buffered reaction mixture containing L-methionine, an electron donor, nitro blue tetrazolium or NBT, a redox indicator, and flavin mononucleotide or FMN, a photosensitizer.
Add this mixture to a tube containing the bacterial pellet and vortex.
Transfer the solution to a glass tube.
Now, irradiate the mixture with violet light to activate FMN, which accepts an electron from L-methionine, forming a reactive intermediate.
This intermediate transfers the electron to dissolved molecular oxygen, generating reactive oxygen species or ROS near the bacterial surface.
The resulting ROS damages cellular components and reduces bacterial viability.
The ROS also reduces NBT to formazan, a blue-colored product that deposits near the bacterial surface.
Transfer the mixture to a tube, centrifuge it, and discard the supernatant.
Resuspend the pellet in an organic solvent to solubilize formazan.
Finally, measure the absorbance to determine light-induced bacterial inactivation through ROS generation.
Prepare the Staphylococcus aureus samples as described earlier to obtain a cell pellet. To 75 milliliters of phosphate buffer add 0.1093 grams of L-methionine, 0.1 gram of NBT, and 25 milliliters of FMN solution.
Add one milliliter of each reactant solution with varying FMN concentrations to the cell pellets and vortex. Irradiate the solutions under violet light at 10 watts per meter square for 10 minutes. Centrifuge the mixture at 14,000 G for 10 minutes and decant the supernatant.
Resuspend the pellet in one milliliter of dimethyl sulfoxide to extract the reduced NBT. Record the absorbance at 560 nanometers.
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