3. Consumables: sterile culture flasks, cryo-tubes or straws, pipettes, boxes, or canisters for storing tubes/straws, respectively.
4. Safety equipment: lab coat, cryo-gloves, protective eyewear,
long forceps, and tweezers (see Note 3).
5. Post-thaw: a water bath (30–35
C) for thawing; either an
inverted microscope for verifying cell viability or a fluorescent
microscope if using a vital stain.
6. Appropriate sterile culture media for incubation before and
after cryopreservation and to prepare cryoprotective solutions
(e.g., dimethyl sulfoxide, glycerol, sorbitol, polyvinylpyrrolidone (alone and in mixtures)).
3 Methods
3.1 Standard Method
for Cryopreserving
Microalgae (Used
at the RCC and CCAP)
Cultures should be in late exponential or early stationary phase (i.e.,
dense but healthy) when harvested for cryopreservation. All culture
handling should be conducted in a laminar flow cabinet, using
sterile consumables and sterile culture medium (the same medium
used for routine maintenance of the strain). It is very important to
record detailed and accurate information throughout the cryopreservation process, and the use of cold-resistant printed labels on all
cryo-tubes is recommended.
1. Concentrate the algae by decantation or by gentle centrifugation to obtain the highest possible density.
2. Cryopreserve using 10% (v/v) dimethyl sulfoxide (DMSO):
add 1 mL of concentrated DMSO to 9 mL of culture in a
sterile Falcon tube (maintain at the same temperature as the
original culture and at low light intensity). Alternatively prepare a 20% (v/v) DMSO solution in sterile culture medium and
dilute 1:1 with the culture in a sterile Falcon tube.
3. Allow the mixture to equilibrate for 5 min, and then aseptically
put 1 mL into each cryo-tube (total equilibration time
10–15 min).
4. Load cryo-tubes into the controlled rate freezer (see Note 4)
and initiate the following program: starting temperature equivalent to temperature at which original culture is maintained,
cooling at 1
C/min down to À40
C, hold at À40
C for
5 min.
5. Transfer cryo-tubes directly into a recipient containing liquid
nitrogen taking appropriate safety precautions (see Note 5).
6. Transfer tubes into plastic boxes for storage in liquid nitrogen
and/or À150
C freezer.
7. For thawing, transfer the cryo-tubes from the liquid nitrogen
or À150
C freezer to a warm water bath (30
C) and leave
Cryopreservation of Algae
609
4. Safety equipment: lab coat, cryo-gloves, protective eyewear,
long forceps, and tweezers (see Note 3).
5. Post-thaw: a water bath (30–35
C) for thawing; either an
inverted microscope for verifying cell viability or a fluorescent
microscope if using a vital stain.
6. Appropriate sterile culture media for incubation before and
after cryopreservation and to prepare cryoprotective solutions
(e.g., dimethyl sulfoxide, glycerol, sorbitol, polyvinylpyrrolidone (alone and in mixtures)).
3 Methods
3.1 Standard Method
for Cryopreserving
Microalgae (Used
at the RCC and CCAP)
Cultures should be in late exponential or early stationary phase (i.e.,
dense but healthy) when harvested for cryopreservation. All culture
handling should be conducted in a laminar flow cabinet, using
sterile consumables and sterile culture medium (the same medium
used for routine maintenance of the strain). It is very important to
record detailed and accurate information throughout the cryopreservation process, and the use of cold-resistant printed labels on all
cryo-tubes is recommended.
1. Concentrate the algae by decantation or by gentle centrifugation to obtain the highest possible density.
2. Cryopreserve using 10% (v/v) dimethyl sulfoxide (DMSO):
add 1 mL of concentrated DMSO to 9 mL of culture in a
sterile Falcon tube (maintain at the same temperature as the
original culture and at low light intensity). Alternatively prepare a 20% (v/v) DMSO solution in sterile culture medium and
dilute 1:1 with the culture in a sterile Falcon tube.
3. Allow the mixture to equilibrate for 5 min, and then aseptically
put 1 mL into each cryo-tube (total equilibration time
10–15 min).
4. Load cryo-tubes into the controlled rate freezer (see Note 4)
and initiate the following program: starting temperature equivalent to temperature at which original culture is maintained,
cooling at 1
C/min down to À40
C, hold at À40
C for
5 min.
5. Transfer cryo-tubes directly into a recipient containing liquid
nitrogen taking appropriate safety precautions (see Note 5).
6. Transfer tubes into plastic boxes for storage in liquid nitrogen
and/or À150
C freezer.
7. For thawing, transfer the cryo-tubes from the liquid nitrogen
or À150
C freezer to a warm water bath (30
C) and leave
Cryopreservation of Algae
609