This study is a simple, versatile and easy system to examine different photodynamic effects on various microorganisms and cells. In this system, the LED can be fit into different arrangement depending on experiment design. Different condition of PDT can be computated in a 96-well plate or even a 384-well plate in one experiment.
This technique can be used to treat fungal keratitis because of which globally, around 150, 000 people lose their eyes in spite of intensive treatments. To begin, cut four green LEDs from an LED strip and align them with three wells of a 96-well plate. Measure the fluence rate of the LED at 540 nanometers with a light power meter.
Maintain constant temperature at 25 degrees Celsius with an electric fan that was assembled alongside the plate during irradiation. With a sterile loop, pick a single colony of Candida albicans from an agar plate and add it to a sterilized glass tube containing three milliliters of YPD medium. Incubate the tube at 25 degrees Celsius at a rotation speed of 155 RPM for 14 to 16 hours to grow the yeast culture.
Next dilute the overnight culture with YPD medium to an OD 600 value of 0.5. Incubate at 30 degrees Celsius with rotation at 155 RPM for four hours to achieve the log growth phase. Repeat the dilution of the log phase culture with fresh YPD medium to an OD 600 value of 0.65, to get approximately one times 10 to the seventh colony forming units per milliliter.
Simultaneously prepare a 4%stock solution of rose bengal in 1X PBS. Filter sterilize with a 0.22 micrometer filter. Add 111 microliters of 2%rose bengal solution to one milliliter of log phase culture, and co-culture at different time points at room temperature to understand the absorption of RB in the cells.
Centrifuge at 16, 100 times G for 2 1/2 minutes at room temperature, and wash the co-culture three times with one milliliter of 1X PBS. After washing, resuspend the Candida albicans culture in one milliliter of 1X PBS. For each condition, distribute the culture into three different wells in a 96-well plate.
Align the wells with the LED array. In the light exposed groups, turn on the electric fan and light. After irradiation, perform tenfold serial dilutions twice by taking 20 microliters of the co-culture solution from one well, and adding it to a tube containing 180 microliters of 1X PBS.
Then drop 20 microliters of each serial dilution in one quadrant of a YPD agar plate to obtain countable colonies on the plate. Fluorescent microscopy showed immediate staining of Candida albicans with rose bengal under red fluorescence. After 15 minutes of incubation, most of the cells were stained with rose bengal, indicating that it enters the cells in a time-dependent manner.
Further, activation of rose bengal with light generates free radicals and singlet oxygen in the cells, resulting in cell death. In absence of rose bengal or irradiation, no cell death was observed. Candida albicans was inhibited after green light irradiation in the presence of 0.2%rose bengal in a light dose-dependent manner.
It is important that first the 96-well plate must be aligned with the center of the LED. Second, the agar plate used must be dried thoroughly to prevent the mixing of separate colonies. The Candida albicans that grew on the plate can be further sent for gene analysis, which can answer how PDT affects gene expression of the cells.
These techniques pave the way to examine how PDT works on different cell types, either cancer cells, bacteria, fungi or viruses with an easy, inexpensive and versatile platform.
