9°C and from 0 to 182 m, respectively. For soil samples, sterile 50 ml tubes were filled with soil, sealed and stored at −20°C. For water samples, 200–500 ml of water were collected from terrestrial sources and processed in situ using the 55-PLUS™ MONITOR system (Millipore, Billerica, MA, USA,) with cellulose filter for yeasts and molds, as specified by the manufacturer. The dishes were then stored at 4°C until processing. Figure 1 A. Sample site locations on King Selleck VX-661 George Island. B – E, Zoomed-in details of the principal sampling zones. Collection sites
of soil and water samples are marked with T# and H#, respectively. Sample processing, yeast cultivation and isolation Five grams of each soil sample was suspended in 5 ml selleck screening library of sterile water by vigorous agitation on a vortex for 10 min. Following decantation of the coarse particulate material, 200 μl of the suspension was seeded onto plates containing YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone) supplemented with 2% glucose and 100 μg/ml chloramphenicol (YM-cm). The plates were incubated at 4, 10, 15 and 22°C. Duplicate of water sampling dishes were incubated at 4 and 10°C. The plates were incubated for 3
months and periodically inspected for colony development. Once a colony became visible, it was immediately transferred to fresh YM-cm plates and incubated at the same temperature as the source-plate. The procedure was repeated for each soil sample to maximize the number of isolates. mafosfamide Long-term preservation of the yeast isolates was achieved via two methods; the gelatin drop
method [42, 43] and cryopreservation at −80°C in 30% glycerol. Determination Compound C order of growth temperatures and carbon source assimilation Yeast growth at different temperatures was assessed by a method based on comparison of colony sizes on solid media, which is applicable to the determination of minimum inhibitory concentration in yeasts . The yeasts were seeded onto YM plates, incubated at 4, 10, 15, 22, 30 and 37°C, and the colony sizes were recorded daily. For each yeast at each temperature, a plot of colony size vs. incubation time was constructed; the temperatures at which colony diameter increased significantly were considered as positive for growth, while the temperature at which the slope changed most rapidly was considered as the “best” or “optimal” for the growth. Glucose fermentation test were performed using a Durham tube. The assimilation of 29 different carbon sources was determined using the API ID 32C gallery (bioMérieux, Lyon, France) as specified by the manufacturer. Briefly, a colony portion was suspended in 400 μl of sterile water. Following adjustment to A600nm≈0.5 (equivalent to 2 McFarland standard), 250 μl of the suspension was added to an ampule of api C medium. Each well of the strip was seeded with 135 μl of this final suspension and incubated in a humid chamber.