sion of the air bubble measured with a stage micrometer and microscope. In artificial pond water of 0.001 osmolal, turgor pressure is typically between 7 and 8 atm and diminishes linearly to zero as extracellular osmolality is increased to the isotonic value of 0.320-0.370. Further increases in osmolality result in the progressive development of a negative pressure that reaches about -0.05 at 0.700 osmolal. This finding supports Meryman’s “minimum cell volume” hypothesis and demonstrates a force that could be the agent of damage to the cell membrane by hypertonic solutions. (Supported in part by Office of Naval Research Contract N00014-69-C-0162.) 16. Hyperbaric Blood Freezing. FREDERIC AHLGREN,* FRANK DORMAN* AND PERRY L. BLACKSHEAR, JR.* (Department of Mechanical Engi-
neering, University of Minnesota, Minneapolis, Minnesota 55455). Introduced by E. F. Graham. This paper reports on the experimental determination of low hemolysis blood freezing using hyperbaric pressure and controlled cooling rates. A test apparatus has been constructed which is capable of freezing blood at controlled rates at pressures of up to 2000 atm. The blood is held in a 0.5-mm thick annulus region and can be cooled to a temperature of -60°C at rates between 1 and 2OO”C/min. Whole dog blood and blood with various amounts of glycerol were pressurized at +4”C to a test pressure and then frozen at a controlled rate. The samples were then thawed quickly at the same pressure and the level of hemolysis determined. It has been found that pressure allows one to freeze whole blood at rates below lOO”C/min and obtain about 30% recovery. It has also been found that the effects of glycerol and pressure are additive so that low levels of hemolysis can be obtained at rates below lO”C/min. The theory that freeze damage can be reduced by conditions that produce breaking of the icelike structure of water is further reinforced by the equivalent effect of high pressure and glycerol. 17. Freezing
Under Gaseous Pressure: Efiect on Cells and Mechanism. GERHARD J. HAAS AND HENRY E. PRESCOTT,JR.* (Technical Center, Gen-
eral Foods Corp., White 10625).
When cellular materials are first saturated with gas under pressure and subsequently frozen under gaseous pressure, much of the gas is retained even upon thawing at atmospheric pressure. This effect is modified by the type of gas used to pressurize the system and by the type of biological material.
Only certain gases such as nitrogen and methane are effective, while others such as carbon dioxide and nitrous oxide are not. Gases which pass through artificial membranes quickly are usually less effective than those which pass through slowly. Pressure frozen substances have more opacity than ordinarily frozen ones, probably because of entrapped gas bubbles. After thawing, numerous gas bubbles are easily visible microscopically. If the material is then air-dried, the gas remains in the tissue and prevents shrivelling. Certain cells are much more capable than others in retaining gases, and some such as mushroom (Agaricus) hyphae will not retain gas. Pressure freezing-air drying offers an alternative to freeze drying for the preservation of tissues without shrivelling. 18. Cryopreservation Under High Hydrostatic Pressure. MAXIM D. PERSIDSKY (The Institute of Medical Sciences, Pacific Medical Center, Clay and Webster Streets, San Francisco, California 94115). Use of high hydrostatic pressure for controlling phase transition in water may offer new opportunity for improving cryopreservation procedures. Further development by this author of the theoretical background on the thermodynamic events in water during phase transition revealed, among other facts, that: (1) ice I may be formed at pressures up to 40,000 psi and temperatures below -26°C; (2) above 31,000 psi ice I is unstable and is transformed into ice III; (3) ice III can be induced either by rapid pressure reduction or by rapid pressure increase; and (4) a new transient form of high pressure ice may exist. Based on this new information it was possible to formulate key experimental conditions necessary for exploration of cryobiological potentials of the use of high pressure. These conditions are: (1) spontaneous freezing under high pressure; (2) induced freezing by one step-pressure reduction; (3) induced freezing by multiple step-pressure reduction; (4) induced freezing by pulsating multiple step-pressure reduction; and (5) induction of ice III by rapid pressure increase. Results of EM examination of heart valve tissue following treatment under each of these conditions revealed that freezing induced by one step-pressure reduction yielded viable cells, as judged by their morphology. This and other results will be presented. (Supported by National Institutes of Health Grant 5 R01 HE12668-02.) 19. Ultrastructural Cryoinjury of Isolated Mitochondria from the Renal Cortex. J. K. SHERMAN