Sabouraud dextrose agar plates

Whole corneas from the murine model of Aspergillus fumigatus-induced fungal keratitis were homogenized in 1 ml of sterile PBS. Subsequently, serial 10-fold dilutions were performed and plated onto Sabouraud dextrose agar plates (Merck, Darmstadt, Germany). The plates were then incubated at 37°C for 24 h, and the CFU number was determined by direct counting.

All Candida isolates obtained from patients with signs and symptoms of fungal infections were included in the present study. During the study period from Dec 2019 to Dec 2021, Candida isolates were collected from clinical samples of patients admitted to 10 tertiary care Medical University Hospitals in Iran (i.e., Shiraz, Ahvaz, Isfahan, Kerman, Mashhad, Khorram Abad, Sanandaj, Urmia, Yasuj, and Zahedan). Clinical samples (i.e., sinuses, lung tissue, blood, oral lesions, pleural tap, bronchoalveolar lavage, sputum, vagina, and cutaneous samples) were cultured on Sabouraud dextrose agar plates (Merck, Germany) and incubated at 22 to 25°C for 3 to 5 days.

Clinical samples (n = 3,500) from patients and environmental swabs from different wards of 11 Iranian hospitals were cultured on Sabouraud dextrose agar (SDA) plates (Merck, Germany) and incubated at room temperature for 7 to 10 days. The Aspergillus species were identified to the species complex level based on colony morphology, lactophenol cotton blue microscopy, PCR-restriction fragment length polymorphism (RFLP), and sequencing of the beta-tubulin gene. For DNA extraction, the isolated species were grown in Sabouraud dextrose broth (Merck, Darmstadt, Germany) for 2 to 3 days at 30°C and 120 rpm. DNA was extracted from young hyphae using the phenol-chloroform method. PCR amplification of the beta-tubulin gene was done using forward primer 5′-GGT AAC CAA ATC GGT GCT GCT TTC-3′ and reverse primer 5′-ACC CTC AGT GTG ACC CTT GGC-3′ (38 (link)). The PCR products were digested with a single AlwI restriction enzyme. Fifty isolated species were subjected to DNA sequencing. The data were analyzed with the NCBI nucleotide database (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Clinical samples (n = 3,500) from patients and environmental swabs from different wards of 11 Iranian hospitals were cultured on Sabouraud dextrose agar (SDA) plates (Merck, Germany) and incubated at room temperature for 7 to 10 days. The Aspergillus species were identified to the species complex level based on colony morphology, lactophenol cotton blue microscopy, PCR-restriction fragment length polymorphism (RFLP), and sequencing of the beta-tubulin gene. For DNA extraction, the isolated species were grown in Sabouraud dextrose broth (Merck, Darmstadt, Germany) for 2 to 3 days at 30°C and 120 rpm. DNA was extracted from young hyphae using the phenol-chloroform method. PCR amplification of the beta-tubulin gene was done using forward primer 5′-GGT AAC CAA ATC GGT GCT GCT TTC-3′ and reverse primer 5′-ACC CTC AGT GTG ACC CTT GGC-3′ (38 (link)). The PCR products were digested with a single AlwI restriction enzyme. Fifty isolated species were subjected to DNA sequencing. The data were analyzed with the NCBI nucleotide database (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Fungal and mammalian cells were grown as described above. The cells were washed 3 times in either sterile 1x PBS or RPMI-1640 and in the following conditions: 515 G (for mammalian cells) or 3000 G (for fungal cells) for 5 min and at 4 °C. Following the washes the cells were counted using a hemocytometer. Mammalian cells were then incubated in the presence of isotype control or TIGIT-blocking antibodies (1 μg/10x⁵ cells, diluted in RPMI-1640-based growth media described above) for 1 h on ice. Following the blocking stage, the effector mammalian cells were mixed with the target fungal cells in U-shaped 96-well plates (5 × 10⁴ mammalian cells, 1000 fungal cells) and in a final volume of 200 μl RPMI-1640-growth media (described above). The cells were co-incubated for 12–14 h in a stationary 37 °C, 5% CO₂ incubator and then serially diluted in 1xPBS and plated on Sabouraud dextrose agar plates (Sigma-Aldrich). The plates were incubated in a stationary 30 °C incubator and the number of colonies in each plate was measured after 24–48 h. The % Colony Forming Units (CFU) reduction was calculated by comparing the CFU counted in each Sabouraud dextrose agar plate to the CFU measured in a control plate in which a culture identical to all other samples but lacking effector mammalian cells was plated.