Ion concentrations in the contractile vacuole (CV) and cytoplasm of Acanthamoeba castellanii were measured by analytical electron microscopy to investigate how these cells utilize ion transport for their osmoregulation. Electron energy-loss spectroscopy (EELS) of frozen-hydrated sections yielded the CV water volume; digital dark-field imaging of dried sections in the scanning transmission electron microscope (STEM) provided the mass of salts. These combined data gave estimated vacuolar osmolarities of 220+/-24 mosmoles for cells cultured in 100% growth medium (212+/-10 mosmoles) and 180+/-20 mosmoles for cells cultured in hypotonic medium (60+/-10 mosmoles). Energy-dispersive X-ray spectroscopy (EDXS) analysis of the salts in the contractile vacuole revealed high vacuolar concentrations of both Na+ and K+, with the ratio of these ions depending on the osmolarity of the extracellular medium. High [Na+] found in contractile vacuoles of amoebae cultured in 100% growth medium suggests that the vacuolar membrane contains an H+/Na+ exchanger in addition to an H+ pump. Amoebae cultured in hypotonic media appear to osmoregulate by using K+ as well as Na+. Low [Cl-] found in the vacuolar fluid suggests that HCO3- is the major anion, and indicates that a Cl-/HCO3- exchanger might also be present.
