title: Use of FM1-43, a membrane-specific fluorescent dye, to estimate plasma membrane integrity in the cryopreservation of green algae.
abstract: 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.
  
  
  METHODS
  We used the two-step freezing method for the cryopreservation of the green alga Parachlorella kessleri.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: T
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba, Japan.
authors.forename: C
authors.initials: C
authors.lastname: Hirai
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba, Japan. JST, CREST, Kashiwanoha, Kashiwa, Chiba, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Ota
authors.affiliation: Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, Nagasaki , Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kuwano
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba, Japan. JST, CREST, Kashiwanoha, Kashiwa, Chiba, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kuwano
mesh_terms: D002462:Cell Membrane / Q000737:chemistry; D000460:Chlorophyta / Q000737:chemistry / Q000166:cytology*; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D019855:Ethylene Glycol / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis*; D005615:Freezing; D011726:Pyridinium Compounds / Q000032:analysis*; D000644:Quaternary Ammonium Compounds / Q000032:analysis*
chemical_list: D003451:Cryoprotective Agents; C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide