1. 1. A new and accurate method is described for carrying out osmotic hemolysis at different rates. The method requires a variable-speed syringe pump. The NaCl concentration, the relative tonicity, T, and the index of the membrane-straining rate, (-1/T2)·(dT/dt), can be precisely known for any time point during hemolysis. 2. 2. The critical hemolytic volume, Vc, obtained by the Guest and Wing method, was found to be independent of the membrane-strain rate and independent of the temperature. This finding is consistent with the hypothesis of Rand that the membrane hemolyzes at the same strain. 3. 3. The osmotic fragility was reduced at higher temperatures and at slower hemolyzing rates. Since Vc was independent of temperature and strain rate, a shift in osmotic fragility did not necessarily mean that the Vc was larger. 4. 4. The prelytic release of K+ in fast hypotonic hemolysis amounted to 10 % of the intracellular K+. This increased to about 20 % for slow hemolysis. A prelytic loss of intracellular K+ quantitatively explains the reduced osmotic fragility observed in gradual hemolysis; a prelytic loss of K+ most probably also accounts for the reduced fragility at elevated temperatures. 5. 5. The time course of the changes in mean cell volume that a population of erythrocytes undergo in osmotic hemolysis to become spherical ghosts was monitored by a Coulter counter and computer. In 0.30 % NaCl the mean cell volume became 120-125 μ3 within 10 sec, then remained unchanged for 30 sec (or sometimes dropped transiently by 5 μ3), and the mean cell volume of the spherical ghosts reached a final value of about 152 μ3. These results are compatible with the idea that individual erythrocytes hemolyze at approximately the same Vc (of about 150 μ3) but at different times (so that the critical mean cell volume is less than the Vc). 6. 6. The volumes of cells in isotonic solution were unchanged over a wide range of temperature. Considering the observations that the Guest and Wing Vc was independent of temperature and the Hoffman Vs (sealing volume) varied markedly with temperature, the facts indicate either that (a) there was a transient collapse of the ghost before sealing, or (b) membrane sealing occurred before transmembrane equilibration of hemoglobin. The sealing volume, Vs, does not necessarily, therefore, have the same value as Vc. © 1969.
