61 CryoLetters 28(1), 61-68 (2007)  CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK RECRYSTALLIZATION INHIBITION ASSESSED BY SPLAT COOLING AND OPTICAL RECRYSTALLOMETRY David A. Wharton 1, * , Peter W. Wilson 2 , Jodi S. Mutch 3 , Craig J. Marshall 3 and Miang Lim 4 Departments of Zoology 1 , Physics 2 , Biochemistry 3 and Food Science 4 , University of Otago, P.O. Box 56, Dunedin, New Zealand. *Corresponding author - david.wharton@stonebow.otago.ac.nz Abstract Levels of recrystallization have been measured by two distinct techniques; a splat cooling assay and a new device, an optical recrystallometer, which measures the change in light transmittance through a frozen sample. Both techniques indicate the presence of recrystallization inhibitors in a grass extract and in other samples. The advantages of each method of measuring recrystallization are discussed. Keywords: ice, recrystallization, splat freezing, antifreeze protein. INTRODUCTION Frozen aqueous solutions tend to recrystallize at high subzero temperatures, unless recrystallization inhibitors are present. This phenomenon is familiar to anyone who has let their ice cream almost melt and then put it back in the freezer. It becomes grainy due to the larger ice crystals, which have grown at the expense of smaller ones. Because of the Kelvin Effect the smaller ice crystals, which have high curvature, have a lower melting point, and melt at a higher annealing temperature. The liquid water released then migrates to a nearby (and probably larger) ice crystal and refreezes, as this crystal has a higher melting point, which is above the ambient temperature in the matrix (7). The overall effect is that a matrix held at a high subzero temperature develops fewer but larger crystals with time. Recrystallization may also occur at a constant temperature and in a completely-frozen solution due to variations in curvature between adjacent ice crystals that produce differences in internal pressure and lead to grain boundary migration (7). Proteins that affect the formation and stability of ice (ice-active proteins, 18) may either initiate ice formation (ice nucleating proteins), inhibit ice formation (antifreeze proteins) or show little or no thermal hysteresis (inhibition of ice crystal growth in the presence of an ice crystal, 3) but inhibit recrystallization (recrystallization-inhibiting proteins, RIPs, 18). RIPs and antifreeze proteins, even in small quantities, are very good at inhibiting recrystallization (6). These classes of proteins are known to bind to the surface of ice crystals (5) and it is thought that this prevents the migration of water molecules and so inhibits recrystallization. Plant, nematode and some arthropod RIPs are very good at recrystallization inhibition (RI), even though there is little or no measurable thermal hysteresis (11,14,16,18).