A new method of freezing-point determination for small quantities.

To a very large extent progress in the field of invertebrate physiology is a measure of progress in the technique of manipulating small structures and analysing small samples. Within this field the study of osmotic regulation is no exception. Krogh (1938) has summarized the known methods of measuring osmotic pressure, applicable to quantities of fluid of the order of O-I-I-O cu.mm. The freezing-point method now to be described is capable of dealing with quantities one thousand times smaller. II. PRINCIPLE The main difficulty of the orthodox freezing-point methods, when applied to small quantities, is the difficulty of avoiding undercooling. Drucker & Schreiner (1913) showed that this could be overcome by first freezing the sample and then warming slowly so as to observe (with a hand lens) the disappearance of the last crystal of ice; the temperature at which this happens is the freezing-point, provided that certain precautions have been taken. This method was used by Fritsche (1916), who determined the freezing-points of 1-3 cu.mm. samples of Daphnia blood to the nearest o-oi°C. As described by Fritsche the method is difficult, requiring care and practice—'die ganze Methode ziemlich individuell ist'—and does not seem to have been used by others. It is apparent, however, that the main difficulty of the method lay in the control of temperature, and that the apparatus was not such as to make temperature control at all easy. It was therefore decided to examine the possibility of developing the method along the lines of better temperature control while retaining the principle of observing the disappearance rather than the formation of ice. In addition, it was proposed to observe the ice-crystals with a microscope, so as to make it possible to work with smaller quantities, and to keep the sample under liquid paraffin, so as to avoid loss of water by evaporation.