Cryobiology 52 (2006) 319–322 www.elsevier.com/locate/ycryo 0011-2240/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2006.01.001 Rapid-cooling and storage of plant nematodes at ¡140°C  Tiziana Irdani ¤ , Beatrice Carletti, Laura Ambrogioni, Pio Federico Roversi CRA-Istituto Sperimentale per la Zoologia Agraria, Cascine del Riccio, 50125 Firenze, Italy Received 11 October 2005; accepted 11 January 2006 Available online 20 February 2006 Abstract Low temperatures can assure the long-term or even indeWnite preservation of important biological specimens. Nema- tode cryopreservation allows for the availability of large numbers of living nematodes at any one time, especially for exper- imental purposes. New isolates of Bursaphelenchus have recently been collected, including Bursaphelenchus eremus (Rühm) Goodey.This species was identiWed in north-central Italy on dying oak trees and from the bark beetle Scolytus intricatus Ratzeburg as dauer larvae. We therefore, sought to develop a cryopreservation technique for the long-term storage of all available Bursaphelenchus spp. The technique consists of a rapid-cooling protocol involving immersion in a liquid nitrogen bath before storage of the frozen samples in a mechanical freezer at ¡140 °C. The survival of nematodes subjected to this rapid-cooling protocol was higher than previously reported using slow-cooling methods and is suitable for several species of Bursaphelenchus and other phytoparasitic nematodes.  2006 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Cryoprotectant; Bursaphelenchus spp.; Long-storage nematode cultures Cryopreservation is a practical means of preserv- ing organisms for long periods. However, diVerent species have distinct requirements for cryopreserva- tion, and the protocols are established empirically case by case. Nematodes, like several other species of parasites and insect embryos, often cannot be cryo- preserved by slow-cooling protocols, but have a requirement for vitriWcation. Yet, even when slow- cooling methods do yield viable organisms, vitriWca- tion often leads to a marked improvement of the survival rate [4]. VitriWcation of aqueous solutions containing high concentrations of solutes, such as ethanediol and sugars, can be achieved by rapid-cooling to below the glass transition temperature (T g ) and then rapid warming to minimize devitriWcation [5]. Thus far, cryopreservation procedures have been explored for the storage of animal parasitic nema- todes and a few plant parasitic nematodes [3,8,2], including two closely related species belonging to the pinewood nematode complex: Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle and Bursaphe- lenchus mucronatus Mamiya et Enda [7,6]. For  This study was supported by a special grant from the Agricul- ture and Forestry Ministry “Storage at low and ultra-low temper- atures of entomopathogenic microrganisms, nematodes and arthropods important in agricultural defence strategies” and by a National Research Program (PREVENTO) “Protection of the environment and agriculture against the risk of importation of exotic arthropods.” * Corresponding author. Fax: +39 55 209177. E-mail address: tiziana.irdani@isza.it (T. Irdani).