4.5. Candida albicans Inhibition Assay

There were 22 test photosensitizers from the ESR assay in the experimental group to test for Candida albicans inhibition. Nystatin (1:100,000 U/mL) oral suspension was set as the positive control and phosphate buffer saline as the negative control.

Candida albicans ATCC 10231 was grown on Sabouraud dextrose broth, then blended in a high-speed blender at 350× g for 10 min, and washed with phosphate buffer saline twice. We then adjusted the density with a spectrophotometer at the wavelength 530 nm at 0.381. The solution containing yeast Candida albicans in the form of 10⁷ cells/mL final concentration was achieved.

Biofilms were produced in 6-well plates based on but modified from Jin et al. [38] and Thein et al. [39], with glass coverslips, and incubated for 1.5 h at 37 °C in an orbital shaker at 75 rpm to promote yeast adherence to the surfaces of the wells (adhesion phase). Then, we added 1× yeast nitrogen base and 50 mM glucose for promoting biofilm growth, and then continued incubating for 48 h to acquire mature Candida biofilm.

We took the glass slide containing the Candida biofilm to the study plate, and added 2 mL of test photosensitizer onto the glass containing the Candida albicans biofilm. We irradiated with the blue dental LED light for 27 s (3 s of irradiation alternately, with 1 s between each session). Upon irradiation completion, this was vibrated with an ultrasonic incubator at 75 rpm for 15 min to separate the unbound colonies. We cultivated a Candida albicans suspension in Sabouraud dextrose agar at 37 °C for 48 h. Then, we counted the colonies of Candida albicans (CFU/mL) by the drop plate technique at 1:1000 dilutions, and a logarithm transformation of CFU/mL was performed.