Types and combinations of cryoprotectant agents

Eight single CPAs commonly used in cryopreserving different types of microalgae were tested empirically to determine the optimal working concentrations^5,30,35 for the three dinoflagellate species. Methanol was preferred to ethanol because it is more effective, penetrates the cells more easily and is less toxic^30,35. The CPAs used were; DMSO (Purity GC ≥ 99.9%, Sigma-Aldrich, Japan), Methanol (MeOH; HPLC grade, ≥ 99.9%, Sigma-Aldrich, Germany), Glycerol (Sigma-Aldrich, Malaysia), Propylene glycol (PG; Sigma-Aldrich, Singapore), Diethylene glycol (DEG; Sigma-Aldrich, USA), Ethylene glycol (EG; spectrophotometric grade, ≥ 99%, Sigma-Aldrich, Malaysia), Polyvinylpyrrolidone (PVP; Sigma-Aldrich, USA) and Polyethylene glycol (PEG; Sigma-Aldrich, Germany). Single CPA solutions were prepared in sterilized growth medium to give final concentrations of 5%, 8%, 10%, 12%, and 15% (v/v or w/v).

For combined CPA treatments, DMSO was preferred because it penetrates the cell membrane and into the intracellular space more easily than glycerol and does not cause excessive bacterial contaminations³⁰. DMSO was combined in a ratio of 1:1 with the non-penetrating compounds proline (Sigma-Aldrich, USA), sucrose (BDH, England), sorbitol (Sigma-Aldrich, USA) and glucose (BDH, England). The concentrations for the combined CPAs were prepared for the different species as shown in Table ​Table11.

Treatment tests using combinations of two cryoprotective agents (CPAs) on the three different dinoflagellate species.