Quantitative Capsule and Melanin Assays

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.