The calculated freezing point depression of freshly excised boiled mammalian tissue is approximately the same as that of plasma. The boiling procedure was chosen to eliminate the influence of metabolism on the level of the freezing point depression. Problems created by the boiling, such as equilibrium between tissue and diluent, change in activity coefficient by dilution, and loss of CO 2 content, are discussed. A frozen crushed tissue homogenate is hypertonic to plasma. Boiling and dilution of such hypertonic homogenate exposed to room temperature for 5 to 15 minutes did not produce significant or unexplicable decreases in its osmotic activity. Moreover, freezing and crushing of a boiled diluted tissue did not produce any increase of the isoosmotic level of freezing point depression. It is possible to explain these data either with the hypothesis of hypertonic cell fluid or with that of isotonic cell fluid. In the case of an assumed isotonic cell fluid, data can be explained with one assumption, experimentally backed. In the case of an assumed hypertonic theory data can be explained only with the help of at least three ad hoc postulates. The data support the validity of the classical concept which holds that cell fluid is isotonic to extracellular fluid.

The freezing point depression of freshly excised frozen tissues, pulverized in a hydraulic press or in a mortar, is greater than that of plasma. Even at 0°C. the freezing point depression of such homogenates increases significantly with time. Dilution data indicate that such freezing point data are valid. The presence of intact cells has been shown in smears of tissues pulverized in a mortar, but not in smears of those crushed in a hydraulic press. The osmolarity of various diluent solutions affects the calculated osmotic activity of tissue homogenates presumably because of delayed diffusion between the diluent and cell fluid. With a hypertonic NaCl diluent, spuriously low values of tissue osmotic activity are found from calculations assuming instantaneous mixing between homogenates and diluents. The limitations of data from cryoscopic experiments and from tissue-swelling experiments are discussed in relation to the basic question of whether or not cell fluid is isotonic to extracellular fluid.