Figure 5. Passive settling times for C. neoformans and C. gattii are strain-specific.

C. neoformans strains H99 and cap59, and C. gattii strains WM161 and 409, were grown overnight in liquid YPD culture, resuspended in cuvettes, and allowed to passively settle while photographs were taken at intervals between 5 min and 6 h, and then again at 26.5 h. Results of A-C represent two independent experiments. A) The cap59 acapsular mutant settled most rapidly, with all cells settled after 60 min. Strains WM161 and 409 settled slower than H99, and even when fully settled, exhibited two distinct layers. B) Based on linear regression, the rate of passive settling was significantly different between strains (P<0.0001). C) Cells from the upper layer of a settled strain 409 culture had significantly larger capsule radii (P<0.0001) and capsule:body volume ratio (P<0.0001) compared to cells from the lower layer. D) Attempts to perform microscopy of the upper layer of a settled strain 409 culture using standard protocols for India ink counterstaining revealed marked clumping of material with cells with large capsules entrapped inside. Scale bar = 20 μm. E) Using a phenol-sulfuric acid assay, the polysaccharide concentration of a confluent strain 409 culture was quantified and compared to that of the upper layer of a settled strain 409 culture, demonstrating that passive settling significantly enriched polysaccharide content in this upper layer.