Figure 3. Experimental halocline formation results in suspension of cryptococci at the air-water interface.

Specific gravity (SG) is the ratio between the density of a compound and that of pure water at 4°C (1.000 g/cm³) and is proportional to salinity. When two liquids are layered, the difference in specific gravity (ΔSG) determines if layers vertically stratify to form a halocline interface. A) If ΔSG < 0, a halocline forms, as illustrated by the addition of PBS with phenol-red (PR) indicator dye (PBS-PR) onto a column of Pacific Ocean seawater (SW). Even very small differences in SG result in halocline formation, as shown by addition of SW-PR (SG = 1.028) to seawater from a different source (LSW; SG = 1.030). Conversely, when ΔSG ≥ 0, no halocline forms and the liquids rapidly mix. B) Strain H99 was suspended in PBS-PR and added onto cuvettes containing serial concentrations of NaCl, illustrating the impact of ΔSG on the size of the halocline. C) Strains H99, cap59, WM161 and 409 were suspended in PBS, and then layered onto either PBS or SW. When cells were suspended in PBS and added to SW, a halocline interface temporarily trapped cells in the top layer. This effect is seen even in the acapsular cap59 mutant. Conversely, when cells grown under identical conditions were suspended in PBS and added to PBS, the cells dispersed rapidly, demonstrating the marked impact of halocline layer formation on buoyancy. C) When cryptococcal cells of strain WM161 were suspended in PBS and added to a conical tube of SW, cells became trapped in the halocline interface, which remained stable throughout gentle tilting, horizontal movement of the tube, or gentle agitation. The stratification was disrupted only with marked agitation, as observed by the rapid color change of the indicator dye.