Sabouraud dextrose agar sda

In sterile working conditions, scrapings from frozen fecal samples were resuspended in 500 µL sterile phosphate-buffered saline (PBS). After vortexing, 100 µL of each sample was spread onto Sabouraud Dextrose Agar (SDA, Sigma Aldrich) plate and Yeast Potato Dextrose agar (YPD, Sigma Aldrich), both supplemented with 0.05 g L^-1 chloramphenicol. Plates were incubated aerobically at 37 °C for 24–48 h. A maximum of ten morphologically similar colonies were processed per individual for identification. Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS, Bruker Daltonics, Bremen, Germany) was used to identify strains by using the manufacturer’s protocol for extended direct transfer (eDT) and ethanol/formic acid extraction.

One hundred clinical isolates were randomly selected from the above previous positive samples for morphological and molecular analysis. Suspensions of spores or mycelia were inoculated onto Sabouraud dextrose agar (SDA, Sigma Aldrich, Germany) plates and incubated in darkness at 37°C for 5–7 days as previously described (11 (link)). Microscopic mounting slides were prepared using lactic acid and examined by a light microscope (Zeiss Axio Scope A1, Carl Zeiss, Göttingen, Germany). Images were annotated in Adobe Photoshop 2022.

Antimicrobial efficacy of the HA powder samples was evaluated on representative microbial strains, including Streptococcus mutans and Candida albicans (ATCC, USA). This was carried out using agar well diffusion assay with Mueller-Hinton agar (MHA) and Sabouraud Dextrose Agar (SDA) media (Sigma-Aldrich, UK).^{35 (link)}
100 µL of microbial suspension were uniformly disseminated onto the surface of MHA and SDA, with a sterile cotton swab. Wells of 6 mm in diameter were holed with a cork borer in an inoculated agar then filled with 50 µL of the sample in distilled water suspension. The plates were then left for about 1 day at 37°C for Streptococcus mutans and 2 days at 28°C for Candida albicans growth. Ciprofloxacin antibiotic was used as a positive control and sterile distilled water was used as a negative control. The experiments were run in three replicates and following incubation, the antimicrobial activity of the samples was assessed by measuring the diameter of the zone of inhibition that formed around the wells.

Prior to in vitro analysis, AuNP were first sterilized using the Tyndallization method and then used for the experiments. For this purpose, the washed AuNP were incubated in the steam of boiling water bath for 30 min and the microtube was placed at RT for 24 h. This procedure was repeated three times and the sterility of AuNP was checked by spreading a loop of AuNP on the surface of Sabouraud Dextrose Agar (SDA, Sigma Aldrich, Prague, Czech Republic) and Nutrient Agar (NA, Sigma Aldrich, Prague, Czech Republic) plates and incubated at 30 °C and 37 °C for 5 days and 24 h, respectively^{53 (link)}.

The fungal strains used in this study were C. albicans ATCC 10231, C. albicans ATCC 64550, C. parapsilosis ATCC 22019, C. tropicalis ATCC 750, C. tropicalis ATCC 200956 (resistant to azoles and AMB) [35 (link)], C. glabrata LMDM 34 (resistant to echinocandins) [36 (link)], C. metapsilosis MUM 15.12, C. orthopsilosis MUM 17.13, C. lusitaniae MUM 17.08, C. krusei ATCC 6258 (Issatchenkia orientalis ATCC 6258), and C. auris CDC B11903. Additionally, nine clinical isolates were included: C. parapsilosis Synlab 406 (FLC-resistant) and eight C. auris isolates identified by MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) according to Zhao et al. [50 (link)]. These yeasts were cultured on Sabouraud Dextrose Agar (SDA; Sigma-Aldrich, St. Louis, MO, USA) for 24 h at 35 °C.

A total of 129
Candidaisolates were tested. The majority of isolates (n = 90) were obtained from blood cultures and the remainder from urine cultures (n: 29) and deep-site specimens (n = 10). Identification of each isolate was performed using conventional methods (germ tube formation, microscopic morphology in corn meal-Tween 80 agar (CMA) (HiMedia, India) and biochemical analysis API 20C AUX (bioMerieux, France) [8]. All specimens were inoculated on to the Sabouraud Dextrose Agar (SDA) (Sigma-Aldrich, Madrid, Spain) and incubated at 35 °C for 24 h. A germ tube test was performed for classification of
Candidaalbicansand nonalbicans
Candida. Positive germ tubes were further incubated at 45 °C to look for the growth. The strains from SDA were inoculated on CMA for morphological examination of the production of chlamydospores, blastospores, true hyphae, and pseudohyphae. They were also inoculated on to Chrom Agar Candida (HiMedia, India) from SDA; identification was made by color and morphology of the colonies according to the manufacturer’s instructions [9]. Repeated isolates from the same patient were excluded.