335 CryoLetters 25, 335-340 (2004) © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK A SIMPLE ICE NUCLEATION SPECTROMETER David A. Wharton 1, * , Jodi S. Mutch 2 , Peter W. Wilson 3 , Craig J. Marshall 2 and Miang Lim 4 Departments of Zoology 1 , Biochemistry 2 , Physiology 3 and Food Science 4 , University of Otago, P.O. Box 56, Dunedin, New Zealand. * Corresponding author - david.wharton@stonebow.otago.ac.nz Abstract The construction of a simple ice nucleation spectrometer is described. It uses 10 μl droplets loaded into glass capillary tubes which are then inserted into an aluminium holder. Each holder takes six capillary tubes surrounding a central thermocouple. Four holders are placed into a cooling block, cooled by fluid from a programmable refrigerated circulator, and the thermocouples interfaced to a computer to record temperatures. Freezing of each sample is detected by an exotherm on the temperature recording, with 24 samples recorded per run. The spectrometer was tested using deionized water, an extract from a New Zealand alpine cockroach and an extract of lawn grass. The cockroach extract is estimated to contain about 10 3 more nucleators, active at -5°C, than the grass extract. Keywords: ice nucleation spectrometer, exotherm, freezing, Celatoblatta quinquemaculata, grass INTRODUCTION Ice nucleators are substances that trigger ice formation in liquids at temperatures below their melting point by acting as nucleating sites. Biological ice nucleators fulfil various functions in organisms. Some bacteria associated with the surface of plants produce potent ice nucleators, which are thought to help the bacteria gain access to plant tissues via freezing- mediated damage to plant epithelia (6). Some freezing tolerant animals produce ice nucleators that ensure freezing at a high subzero temperature, thus limiting the stresses imposed by freezing. Freezing tolerant insects, for example, produce ice nucleating proteins and lipoproteins in their haemolymph (1, 2, 13). The demonstration of ice nucleation activity can involve determining the freezing point (temperature of crystallisation or supercooling point) of a large number of small droplets (40+) of the test solution to produce a nucleation spectrum, which plots the proportion of frozen droplets against the nucleation temperature (7). The droplets are contained in capillary tubes and placed in a refrigerated bath or they are placed on the surface of a metal plate, cooled by refrigerated fluid or thermoelectrically. These instruments are known as ice nucleation spectrometers. The first instruments of this nature determined the freezing of droplets visually (7, 10, 12). Automated systems have been developed that detect the heat of crystallisation produced by the freezing of a droplet and accumulate these data on a computer