Can anyone please suggest how I can prepare glycerol stocks for preserving pure strains of microalgae?
The microalgae culture and its culture medium is totally different from bacteria or fungi and need not store by glycerol method. so you can store it in test tubes or conical flasks with tight cotton plug for longer period. whenever you need to work on it you just take it out from fridge and put it near light then it starts their multiplication.
fridge V CAN USE but culture should be in solid medium (in Petri plates).
The cryoprotectants is dimethyl sulfoxide (DMSO), sorbitol, glycerol, ethylene glycol (EG), and L-proline. These cryoprotectants were added to distilled water (C. vulgaris C-27 and M-207A7) or seawater (N. oculata and T. tetrathele) either alone or in combination at twice the desired final concentration, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was added at a concentration of 0.01 M for adjustment to pH 6.5 and 7.8, respectively. The resulting additive solutions were gradually added to an equal volume of algal cell suspension over the course of 15 min with cooling in an ice bath. Aliquots (1.5 mL) of the resulting suspensions were dispensed into 2-mL cryogenic vials (Corning Inc, USA), and held for 45 min in an ice bath. Vials were set in a container (Mr. Frosty, Thermo Fisher Scientific Inc., USA) and cooled to −40°C in a deep freezer for 4 h, and then immersed in liquid nitrogen. Thawing was carried out by immersing the vials in a 40°C water bath, and then transferring the vials to an ice bath. In order to remove cryoprotectants, the thawed cell suspensions were gradually diluted with 10 ml of 0.01 M HEPES solution (pH 6.5) for C. vulgaris C-27 and M207A7 or with 0.01 M HEPES seawater solution (pH 7.8) for N. oculata and T. tetrathele. The cells were collected by centrifugation (500×g for 10 min) and resuspended in 10 mL of the above solution (Nakanishi et al., 2012).
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