Methods Microbial strains and culture conditions The C. find more albicans strains used in this study were Can14 and Can37. C. albicans Can14 is a wild-type strain SC5314  and C. albicans Can37 is a fluconazole resistant clinical isolate from a patient with oropharyngeal candidiasis . C. albicans Can37 was identified by growth on Hicrome Candida (Himedia, Munbai, India), germ tube test, clamydospore formation on corn meal agar,
and API20C for sugar assimilation (BioMerieux, Marcy Etoile, France). Susceptibility pattern to fluconazole was determined by the broth microdilution assay according to the Clinical and Laboratory Standards Institute (CLSI). Strains were stored as frozen stocks with 30% glycerol at -80°C and subcultured on YPD agar plates (1% yeast extract, 2% peptone, and 2% dextrose) at 30°C. Strains were routinely grown in YPD liquid medium at 30°C Dinaciclib cell line in a shaking incubator. Fungal inocula preparation C. albicans cells were grown in YPD at 30°C overnight. Cells were collected with centrifugation and washed three times with PBS. Yeast cells were counted using a hemocytometer. The cell number was confirmed by determining colony-forming units per mL (CFU/mL) on YPD plates. Inoculation of G. mellonella with C. albicans strains G. mellonella (Vanderhorst Wholesale, St. Marys, OH, USA) in the final larval stage were stored in the dark and used within 7 days from shipment. buy Danusertib Sixteen
randomly chosen G. mellonella larvae with similar weight and size (250-350 mg) were used per group in all assays. Two control groups were included: one group was inoculated with PBS to observe the killing due to physical Thalidomide trauma, and the other received no injection as a control for general viability. A Hamilton syringe was used to inject 5 μL inoculum aliquots into the hemocoel of each larvae via the last left proleg
containing 106 CFU/larvae of C. albicans cells suspended in PBS. After injection, larvae were incubated in plastic containers at 37°C and monitored for survival daily. Chemicals and photosensitizer Methylene blue (MB, Sigma, St Louis, MO) was used at a final working concentration of 1 mM. The dye was dissolved in distilled and deionized filter sterilized water (ddH2O). For each experiment, a new PS solution was prepared daily. Fluconazole (Sigma-Aldrich, Steinheim, Germany) was dissolved in ddH2O and injected in G. mellonella at a concentration of 14 mg/Kg. Antimicrobial photodynamic therapy The G. mellonella larvae were injected with 10 μL of a 1 mM solution of MB 90 min after the Candida infection and the PS was allowed to disperse for 30 min into the insect body in the dark, prior to the light irradiation. A broad-band non coherent light source (LumaCare, Newport Beach, CA) was used for light delivery. This device was fitted with a 660 ± 15 nm band-pass filter probe that was employed to produce a uniform spot for illumination.