Sabouraud dextrose agar

In the laboratory, the tube containing the sample in 0.3 ml of BHI broth was cultured aerobically on Sabouraud dextrose agar (SDA) (Himedia, India) at 37 °C for 48 h, and the resultant growth, if any, was harvested and pure colonies obtained after subculture for 24 h on Sabouraud agar were then stored in 20% glycerol.
A total of four endodontic isolates of C. albicans from another geographical region (Finland) were used for comparative purposes. These were archival strains kindly donated by Dr. TMT Waltimo to the Oral and Biosciences Laboratory, Faculty of Dentistry, Hong Kong University, Hong Kong) [18 (link)]. These isolates were obtained from subjects with primary endodontic infection who were systemically healthy.
Saliva samples from healthy subjects in the UAE were collected randomly to isolate C. albicans for use as reference, control strains of non-endodontic origin, from the oral commensals. For this purpose, saliva from each volunteer was collected by expectoration into a 10 mL container, and cultured in the laboratory as described above, to obtain pure samples, and stored until use in the experiments. The salivary samples were processed following a protocol of Samaranayake et al. [19 (link)].

The strain C. albicans ATCC 90028 was used in the present study. The test culture was maintained in Sabouraud dextrose agar (SDA; Himedia, Mumbai) and cultured routinely in YEPD (Yeast extract peptone dextrose) broth. For experimental analysis, a loopful of culture was used to inoculate YEPD medium and incubated at 37°C overnight. Prior to each bioassay, the overnight culture was adjusted to 0.8 OD at 600 nm (10⁸ CFU/ml). Spider broth (1% mannitol, 0.2% dipotassium hydrogen phosphate, and 1% nutrient broth) was used in the biofilm assay for augmenting hyphal formation (Muthamil and Pandian, 2016 (link)).

Propolis powder was kindly provided by Bee Product Industry Co., Ltd., Lamphun, Thailand. Sorbitan monostearate (Span 60; SP60), polysorbate 80 (Tween 80; TW80), and cholesterol (CHOL) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents used in this study were of analytical grade, including Sabouraud Dextrose Agar (SDA) (HiMedia, Mumbai, India), Sabouraud Dextrose Broth (SDB) (HiMedia, Mumbai, India), Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA), Kaighn’s modification of Ham’s F12 medium (F-12K) (Caisson laboratories Inc., Smithfield, UT, USA), Chloroform (RCI Labscan, Taipei, Taiwan), Potassium phosphotungstic acid (TED PELLA Inc., Redding, CA, USA), 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) (Bio Basic Inc., Markham, ON, Canada), Calcofluor white (CFW) (Sigma-Aldrich, St. Louis, MO, USA), Nile-red dye (Sigma-Aldrich, St. Louis, MO, USA), ProLong Gold anti-fade reagent (Thermo Fisher Scientific, Waltham, MA, USA), Egg Yolk Tellurite Emulsion (HiMedia, Mumbai, India), FUN-1 (Molecular Probe, Waltham, MA, USA), Concanavalin A (Con A)-Alexa Flour 488 conjugate (Thermo Fisher Scientific, CA, USA), lipopolysaccharide (LPS) (Sigma-Aldrich, MO, USA), and interferon-γ (IFN-γ) (Biolegend, San Diego, CA, USA).

Sabouraud dextrose agar (SDA), Sabouraud dextrose broth (SDB), and peptone were obtained from HiMedia (Mumbai, India). Certified standards of DON and ZEA were obtained from Sigma-Aldrich (Bengaluru, India). Immunoaffinity columns specific for DON and ZEA were procured from Vicam (Waters business, USA). The other chemicals used in the study were analytical grade and obtained from Merck Millipore (Bengaluru, India).

Copper nitrate trihydrate (99.5%, Lobal Chemie), trimesic acid (95%, Sigma-Aldrich), N,N-dimethylformamide (DMF; 99.8%, QRec), ethanol (99.9%, QRec), Sabouraud Dextrose Agar (SDA; Himedia), Sabouraud Dextrose Broth (SDB; Himedia) and Potato Dextrose Agar (PDA; Himedia) were of analytical reagent grade. All chemical compounds were diluted with deionized water (greater than 18 MΩ cm, Millipore).