Yeast Isolation and Identification

Serial dilutions (10⁻¹–10⁻³) were made with peptone water (Oxoid Ltd., Hampshire, United Kingdom). Equal volumes (200 μL) from each dilution were spread over the surface of Dicloran Rose Bengal Chloramphenicol (DRBC) (Merck, Darmstadt, Germany) and CHRomagar Candida™ agar plates (BBL International Inc., London, UK). All the plates were incubated for 48 h at 30 °C. Colony counts were performed on individual plates. Representative yeast colonies were selected and grouped by morphotype, isolated, and conserved using Saboraud Dextrose Agar (Merck) immersed in sterile mineral oil and cryopreserved in glycerol 30% (v/v). A macroscopic evaluation of colonies was performed with a stereoscope (Celestron Lab’s S10-60 Stereo, Celestron, LLC, Torrance, CA, USA) taking account of the shape, edge, surface, appearance, elevation, brightness, and consistency. All the yeasts were evaluated for sugar assimilation using the API 20C AUX system (BioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Several carbohydrates were evaluated, including D-Glucose, Glycerol, 2-keto-Gluconate calcium, L-Arabinose, D-Xylose, Adonitol, Xylitol, D-Galactose, Inositol, D-Sorbitol, Methyl-D-Glucopyranoside, N-Acetyl-Glucosamine, D-Lactose (Bovine), D-Maltose, D-Sucrose, D-Trehalose, D-Melezitose and D-Raffinose [28 (link)]. The galleries were incubated at 30 °C for 72 h in an airtight box containing a small volume of water to create a humid atmosphere. The observation of yeast growth was considered positive. The negative control contained no carbon source, and the positive control contained glucose. The carbohydrate assimilation profile obtained for each tested isolate was interpreted using ApiwebTM software (BioMérieux, reference: 40011).