T Yamazaki1, C Hirai2, S Ota3, K Kuwano4, S Kuwano3. 1. Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba, Japan. JST, CREST, Kashiwanoha, Kashiwa, Chiba, Japan. yamazaki@ib.k.u.-tokyo.ac.jp. 2. Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba, Japan. 3. Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba, Japan. JST, CREST, Kashiwanoha, Kashiwa, Chiba, Japan. 4. Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, Nagasaki , Japan.
Abstract
BACKGROUND AND OBJECTIVE: Cryopreservation of microorganism cultures is an important technology for their use as biological and genetic resources; however, the procedure is complicated and depends on the species. MATERIALS AND METHODS: We used the two-step freezing method for the cryopreservation of the green alga Parachlorella kessleri. RESULTS AND CONCLUSION: The optimal cryoprotectant for cryopreservation was 5% dimethyl sulfoxide plus 5% ethylene glycol. This is different from the optimal cryoprotectant for the closely related species Chlorella vulgaris. Efficient cryopreservation of P. kessleri was achieved using methanol, similar to Chlamydomonas reinhardtii. A membrane-specific fluorescent dye, FM1-43, was applied to estimate plasma membrane integrity. We found that the plasma membrane integrity of P. kessleri cells after freeze-thawing was associated with survivability, suggesting that this is a useful index for the optimization of the first step of the two-step freezing method of cryopreservation.
BACKGROUND AND OBJECTIVE: Cryopreservation of microorganism cultures is an important technology for their use as biological and genetic resources; however, the procedure is complicated and depends on the species. MATERIALS AND METHODS: We used the two-step freezing method for the cryopreservation of the green alga Parachlorella kessleri. RESULTS AND CONCLUSION: The optimal cryoprotectant for cryopreservation was 5% dimethyl sulfoxide plus 5% ethylene glycol. This is different from the optimal cryoprotectant for the closely related species Chlorella vulgaris. Efficient cryopreservation of P. kessleri was achieved using methanol, similar to Chlamydomonas reinhardtii. A membrane-specific fluorescent dye, FM1-43, was applied to estimate plasma membrane integrity. We found that the plasma membrane integrity of P. kessleri cells after freeze-thawing was associated with survivability, suggesting that this is a useful index for the optimization of the first step of the two-step freezing method of cryopreservation.