3.3 Cryopreservation
of Hypersaline
Chlorophyte Strains
of Interest
in Biotechnology:
Dunaliella salina,
D. minuta,
D. tertiolecta, D. velox,
D. viridis,
and D. bioculata
1. The strains cryopreserved are listed in Table 3. Cultures should
be healthy and free from contamination and in the late exponential growth phase (determined by daily cell counts in the
period preceding cryopreservation).
2. Strains should be cryopreserved in the same media in which
they are grown. For these strains, this routinely has 50 g/L of
commercially available sea salts added. All media used should
be sterilized by autoclave or filter sterilization before use. All
work should be conducted in a laminar flow clean cabinet and
all equipment cleaned with 70% ethanol before placing into the
laminar flow cabinet. All plastics used are commercially sterilized, and pipette tips have filters to avoid any contamination
from the pipette.
3. All strains outlined above can be cryopreserved in 10% (v/v)
DMSO as a cryoprotectant. This final concentration is
obtained by making an initial solution of 20% (v/v) DMSO
in growth media. This is then filter sterilized using a 0.22 μm
syringe filter and added to an equal volume of the microalgae
culture. It is important to note that the initial addition of
DMSO to media heats the solution; therefore, adequate time
Table 3
Hypersaline chlorophyte strains of interest in biotechnology: Dunaliella salina, D. minuta,
D. tertiolecta, D. velox, D. viridis, and D. bioculata
Strain number
Dunaliella culture
Strain number
Dunaliella culture
PLY SA-1
D. viridis
PLY DF-8
D. salina
PLY SA-2
D. viridis
PLY DF-11
D. salina
PLY SA-3
D. salina aureus
PLY DF-12
D. salina
PLY SA-4
D. salina aureus
PLY T-32
D. bioculata
PLY SA-5
D. velox
PLY T-34
D. minuta
PLY SA-6
D. velox
PLY T-36
D. salina aureus
PLY DF-15
D. salina rubeus
PLY T-37
D. salina aureus
PLY DF-17
D. salina
PLY T-41
D. salina aureus
PLY SA-7
D. bioculata
PLY T-68
D. salina aureus
PLY DF-1
D. salina
PLY T-74
D. salina
PLY DF-3
D. salina
PLY T-75
D. minuta
PLY DF-4
D. salina
PLY T-76
D. minuta
PLY DF-7
D. salina
PLY T-77
D. minuta
UTEX 999
D. tertiolecta
Cryopreservation of Algae
615
of Hypersaline
Chlorophyte Strains
of Interest
in Biotechnology:
Dunaliella salina,
D. minuta,
D. tertiolecta, D. velox,
D. viridis,
and D. bioculata
1. The strains cryopreserved are listed in Table 3. Cultures should
be healthy and free from contamination and in the late exponential growth phase (determined by daily cell counts in the
period preceding cryopreservation).
2. Strains should be cryopreserved in the same media in which
they are grown. For these strains, this routinely has 50 g/L of
commercially available sea salts added. All media used should
be sterilized by autoclave or filter sterilization before use. All
work should be conducted in a laminar flow clean cabinet and
all equipment cleaned with 70% ethanol before placing into the
laminar flow cabinet. All plastics used are commercially sterilized, and pipette tips have filters to avoid any contamination
from the pipette.
3. All strains outlined above can be cryopreserved in 10% (v/v)
DMSO as a cryoprotectant. This final concentration is
obtained by making an initial solution of 20% (v/v) DMSO
in growth media. This is then filter sterilized using a 0.22 μm
syringe filter and added to an equal volume of the microalgae
culture. It is important to note that the initial addition of
DMSO to media heats the solution; therefore, adequate time
Table 3
Hypersaline chlorophyte strains of interest in biotechnology: Dunaliella salina, D. minuta,
D. tertiolecta, D. velox, D. viridis, and D. bioculata
Strain number
Dunaliella culture
Strain number
Dunaliella culture
PLY SA-1
D. viridis
PLY DF-8
D. salina
PLY SA-2
D. viridis
PLY DF-11
D. salina
PLY SA-3
D. salina aureus
PLY DF-12
D. salina
PLY SA-4
D. salina aureus
PLY T-32
D. bioculata
PLY SA-5
D. velox
PLY T-34
D. minuta
PLY SA-6
D. velox
PLY T-36
D. salina aureus
PLY DF-15
D. salina rubeus
PLY T-37
D. salina aureus
PLY DF-17
D. salina
PLY T-41
D. salina aureus
PLY SA-7
D. bioculata
PLY T-68
D. salina aureus
PLY DF-1
D. salina
PLY T-74
D. salina
PLY DF-3
D. salina
PLY T-75
D. minuta
PLY DF-4
D. salina
PLY T-76
D. minuta
PLY DF-7
D. salina
PLY T-77
D. minuta
UTEX 999
D. tertiolecta
Cryopreservation of Algae
615