Spot insight digital camera

For analyzing mating phenotypes opposite mating type (MATα and MATa) cells were cultured in YPD medium at 30°C for 16 h and equal concentration of cells (10⁷ cells/mL) were mixed, spotted onto V8 mating media (pH 5), and incubated in the dark at room temperature for 1–2 weeks. The filamentous growth was monitored and photographed using an Olympus BX51 microscope equipped with a SPOT Insight digital camera. For the cell fusion assay, the concentration of cells was adjusted to 10⁷ cells/mL with phosphate-buffered saline. Each MATα and MATa strain was mixed in an equal volume, spotted onto a V8 medium, and incubated in the dark at room temperature for 24 h. Then, the cells were scraped, resuspended in 1-mL distilled water, and spread onto YPD medium containing both nourseothricin (100 μg/mL) and G418 (50 μg/mL). The plates were further incubated at 30°C, and the number of colonies was counted. For monitoring the pheromone gene expression, the MATα and KN99a strains were mixed with an equal concentration of cells (10⁸ cells/mL), spread onto the V8 medium, and incubated in the dark at room temperature for 18 or 24 h. Then, cells were scraped, pelleted, frozen in liquid nitrogen, and lyophilized overnight for the total RNA isolation, followed by the northern blot analysis with the specific mating pheromone gene (MFα1)-specific probe.

To perform capsule production assay, cells were cultured in YPD broth at 30 °C overnight, and then spotted onto DME medium and incubated at 37 °C for 2 days. After incubation, the capsules were stained with India ink and visualised with an Olympus BX51 microscope equipped with a SPOT insight digital camera. For quantitative analysis of capsule production, 10% formalin-fixed cells were adjusted to 3 × 10⁸ cells per ml in phosphate-buffered saline (PBS), and injected (50 μl) into microhaematocrit capillary tubes (Kimble Chase) in triplicates. All tubes were placed in an upright vertical position for 3 days to precipitate cells by gravity. The relative packed cell volume was measured with haematocrit capillary tubes by calculating the ratio of the lengths of the packed cell phase to the total phase (cells plus liquid phases), as previously described^{15 (link)}. To examine melanin production, cells were grown overnight in YPD broth at 30 °C, then 5 μl of each culture was spotted on Niger seed media containing 0.1% or 0.2% glucose, incubated at 30 °C and photographed after 3–4 days.