This study investigates the effects of flavin mononucleotide (FMN) as a photosensitizer on bacterial viability. By exposing Staphylococcus aureus to varying concentrations of FMN and light, the research assesses the dose-dependent impact on bacterial survival.
Begin by inoculating a pathogenic bacterium into a tube containing a liquid medium.
Incubate to promote bacterial proliferation, then transfer the culture into fresh tubes.
Spin down the cells into pellets and discard the supernatant.
Resuspend the bacterial pellets in buffer solutions containing increasing concentrations of flavin mononucleotide (FMN), a photosensitizer, and a buffer solution without FMN to serve as a control.
Mix the tube contents thoroughly and transfer the suspensions into glass tubes.
Expose the tubes to visible light to photoactivate FMN, which reacts with molecular oxygen to generate reactive oxygen species (ROS).
The ROS damages bacterial cellular components, thereby reducing bacterial viability.
Transfer the sample aliquots onto solid media, spread them evenly, and incubate to allow surviving viable bacteria to grow into colonies.
Post-incubation, quantify the colonies and calculate the percentage reduction in bacterial viability compared to the control to assess the dose-dependent effect of FMN.
Inoculate a colony of Staphylococcus aureus into 10 milliliters of lysogeny broth in a 15-milliliter screw-capped test tube. Grow the culture at 37 degrees Celsius for 16 hours in a shaker. Transfer 0.5 milliliters of the culture to a 1.5-milliliter centrifuge tube.
Dilute the culture to an optical density of 0.5 at 600 nanometers by adding sterilized water. Then, transfer 0.5 milliliters of the culture to a 1.5-milliliter centrifuge tube. Centrifuge at 14,000 G for 10 minutes and decant the supernatant.
Add one milliliter of FMN buffered solution to the cell pellet and vortex. For irradiated control, add one milliliter of phosphate buffer. Transfer one milliliter of bacterial cell solution containing 30-micromolar FMN and phosphate buffer into glass tubes.
Place them for a radiation under violet light at 10 watts per meter square for 30 minutes. Then, transfer one milliliter of bacterial cell solution containing 30, 60 and 120 micromolar FMN and phosphate buffer in glass tubes. Irradiate with blue light for 120 minutes at 20 watts per meter square.
Also, irradiate a tube containing one milliliter of 120-micromolar FMN for 60 minutes. After irradiation, add 0.2 milliliters of the reaction solution onto a luria agar plate. Spread the solution using an L-shaped glass rod and incubate overnight at 37 degrees Celsius.
On the next day, calculate the viable plate count and inactivation rate of Staphylococcus aureus.
繁體中文
