Biggy agar medium

The native strain DiSVA 705 was cultivated in YPD broth for 24 h at 25 °C in a rotary shaker (200 rpm), then 20 µL of the cell suspension was spread on Sporulation Medium (0.25% yeast extract, 0.1% glucose, 0.98% potassium acetate, 2% agar) [29 (link)] and incubated at 23 °C for at least 5 days. When tetrads were observed, a spot of the culture was resuspended in 45 µL of sterile distilled water containing 5 µl of Zymolyase 100-T (ICN Biomedicals, Inc., Irvine, CA, USA) solution (4 mg/mL of sorbitol 2 M) and incubated at room temperature for 10 min to facilitate the cell wall disruption. Ten tetrads were dissected using a micromanipulator (Singer SMS Manual, Somerset, UK) and the single spores were transferred to new YPD-agar plates and grown at 25 °C for 48 h. The viable spores were then analyzed for their H₂S production, spreading them on BiGGY agar medium (Oxoid Ltd., Cheshire, England) and incubated at 25 °C for 48 h. In this medium, the colonies appear white for those H₂S-negative and brown-black for those H₂S-positive.