This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.
The overall goal of this experiment is to evaluate the anti-microbial effect of five-aminolevulinic acid or ALA-mediated photodynamic therapy on a staphylococcus aureus biofilm. This method can answer key questions in the bacterial biofilm field of how photodynamic cell repair might be used as an alternative treatment for controlling biofilm infections. An advantage of this procedure is that it has the potential to be used for other bacteria with minimal adjustment.
To generate a biofilm in a 96 well plate, first inoculate one minus 80 degrees Celsius ThOD 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 degree 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 two times 10 to the ninth colony forming unit per milliliter concentration. Dilute these bacterial suspensions at a one 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.
To count the number of viable bacteria per group at the end of the phototherapy treatment gently wash each well three times with PBS to remove any non-adherent cells. Then use a pipette tip to scrape the adherent bacteria from the bottom of each well. Pull the cells from each group into one eppendorf tube per experimental condition and pellet the cells by centrifugation.
Re-suspend the pellets in one milliliter of 0.25 percent pancreatin enzyme and PBS for a 90 minute incubation at 37 degrees Celsius, followed by another centrifugation. Re-suspend the pellets in 200 microliters of PBS and make one to 10 serial dilutions of each group, adding five microliters of serially diluted sample onto individual tryptone soya agar plates. Then incubate the plates at 37 degrees Celsius for 16 hours and count the number of bacterial colonies per group.
To assess the biofilms by confocal laser scanning microscopy generate and irradiate the biofilms as demonstrated. Wash the irradiated biofilms with PBS and stain the live and dead cells with one milliliter of a green fluorescent nuclear and chromosome procaryotic cell membrane permeable dye and one milliliter of one micromolar propidium iodide, respectively. After 20 minutes remove the excess stain and image the biofilms by confocal laser scanning microscopy under a 63 times 1.4 NA oil immersion objective lens.
Biofilm bacteria viability decreases after ALA phototherapy double treatment compared to control-single or non-treated biofilm bacterial viability and all four strains tested. Visualization of the biofilms by confocal laser scanning microscopy confirms this finding, with most of the cells positive for propidium iodide standing observed in the ALA phototherapy double treatment group. While attempting this procedure it is important to remember that the entire ALA treated bacteria manipulation should be performed in darkness, and the material biofilm should be handled gently to avoid disturbing the formed biofilm.
This message can be applied to explore the antimicrobial effect of photodynamic therapy on bacterial biofilm in vitro by the researchers in the field of bacterial infection field.
