1. Methods for Cryopreservation of Some Species of the Food Microalgae (NRIA Culture Collection)

Abstract

Cryopreservation of five marine microalgae (NRIA strains; Chaetoceros ceratosporum, Chaetoceros gracilis, Chaetoceros simplex, Phaeodactylum tricornutum, Isochrysis galbana) which are widely used in the aquaculture industry as food organisms by the two-step cooling method was attempted. The solution A (50%seawater + 5 %DMSO +0.5M sorbitol) for P. tricornutum and solution B (100%seawater +10%glycerol +0.5M sorbitol) for C. ceratosporum, C. gracilis, C. simplex and I. galbana were employed as cryoprotective solutions in the present study. The cells from a anaexponential phase of growth in P. tricornutum, the cells from a pro-stationary phase in C. gracilis and I. galbana and the cells from a meta-stationary phase in C. ceratosporum and C. simplex were found to be resistant to stresses of freezing and thawing, respectively. The appropriate Cryopreservation procedures were as follows: The cryoprotectants were added gradually over a period of 15 min followed by an equilibration period of 45 min. The cells were then prefrozen to -40℃ at 0.1-1.0/min prior to immersion in LN_2 After storage in LN_2, the cell suspension was thawed quickly by agitation of the vial in a water bath at 40℃, and the cryoprotectants were washed off by gradual dilution with seawater. The best survivals evaluated by the MPN-method were 18.0% in C. ceratosporum, 25.5% in C. gracilis, 21.2% in C. simplex, 40.5% in P. tricornutum and 13.8% in I. galbana, respectively. These survivals were independent of the period of storages at least 150 days.