First attempts to cryopreserve strains from the Coimbra Collection of Algae (ACOI) and the use of image analysis to assess viability
Osório, Hugo C.; Laranjeiro, Nuno C.; Santos, Lília M.A. Santos
Serial sub-culture has always been the principle method for maintaining the cultures of the Coimbra Collection of Algae (ACOI). This technique is time consuming, material and human resources demanding, involves the risk of strain loss and could potentially lead to genetic changes over time. The preservation of cultures at ultra-low temperatures has recently become possible at Coimbra due to the acquisition and establishment of a cryopreservation unit under the ongoing European funded COBRA project (http://www. cobra. ac. uk ). About 100 ACOI strains have been frozen using a standard two-step protocol. The protocol employed dimethyl sulphoxide (DMSO), glycerol and methanol as cryoprotectants in an uncontrolled 2-step cooling process, followed by plunging into liquid nitrogen. The majority of the strains cryopreserved belong to the Chlorococcales, although representatives of the Cyanobacteria, Eustigmatophyceae, Glaucophyceae, Volvocales, Tetrasporales, Chaetophorales, Trebouxiophyceae and Desmidiales were also tested. Some strains within the Chlorococcales and the Eustigmatophyceae demonstrated 100% viability after the treatment; however, no viability was observed for the Tetrasporales and Desmidiales species examined. Viabilities of some strains were compared after cryopreservation in the presence of the three different cryoprotectants and employing a range of intermediate freeze temperatures. DMSO and methanol were the most frequently used cryoprotectants with samples cooled to an intermediate temperature at -80°C. In order to assess strain viability, a new approach based on image analysis of algal colony counts on agar plates was developed. From the data obtained it appears that each strain has its own unique optimal freezing condition, and even closely related strains have different optimum cryopreservation conditions.