should be allowed for the solution to return to room temperature before adding to the cell culture.
4. One milliliter of this culture/cryoprotectant mixture is then
aliquoted into 1.8 mL cryogenic vials and placed into a passive
freezing container (see Note 6) to freeze at a rate of 1
C/min
(we would recommend freezing several vials so that viability
can be tested by thawing while some samples still remain frozen
for long-term storage). This should then remain at room temperature for 10 min in order for the cryoprotectant to penetrate the cells. The passive freezing container is then placed into
a À80
C freezer for 100 min.
5. After this time, the container is removed, and the cryovials
plunged individually into liquid nitrogen (see Note 5) using
large forceps and personal protective equipment (PPE). The
vials are then transferred either into a À150
C ultra-freezer or
into liquid nitrogen storage.
6. When thawing the algae, it is important to expose the samples
to as little light as possible. To thaw, the vials are removed from
the freezer/liquid nitrogen and agitated gently, one at a time, in
a 40
C water bath until all ice crystals are gone (no longer than
2 min) (see Note 7). The thawed vial is then quickly cleaned with
70% ethanol and transferred to the laminar flow cabinet. The
contents are then transferred, by pipetting, into 20 mL of
growth media in a sterile growth flask with a vented lid.
7. This is then placed into a dark box and placed at growth
temperature. The cultures are left in the dark for 24 h. After
this time, the lid is gradually opened to allow some light
in. After 48 h of thawing, the cultures can be removed and
placed at the light intensity they are normally grown at.
8. Viability should be assessed microscopically although it is
worth noting that it can take up to 6 weeks for a strong culture
to establish.
3.4 Cryopreservation
of Macroalgae
In the Culture Collection of Algae and Protozoa (CCAP) laboratory in recent years, a range of macroalgal species have successfully
cryopreserved using slight modifications of the standard two-step
method used for microalgae (see Table 4 and Fig. 2). To successfully
cryopreserve macroalgae, the starting material must be relatively
dense, healthy, and growing well with minimal bacterial contamination. Large thalli and clumps of filamentous species should be
carefully dissected into small pieces, taking care not to introduce
contamination [14–16]. In some cases, the addition of an extracellular membrane impermeable cryoprotectant is required such as
sorbitol or polyvinylpyrrolidone (PVP).
616
Estefania Paredes et al.
4. One milliliter of this culture/cryoprotectant mixture is then
aliquoted into 1.8 mL cryogenic vials and placed into a passive
freezing container (see Note 6) to freeze at a rate of 1
C/min
(we would recommend freezing several vials so that viability
can be tested by thawing while some samples still remain frozen
for long-term storage). This should then remain at room temperature for 10 min in order for the cryoprotectant to penetrate the cells. The passive freezing container is then placed into
a À80
C freezer for 100 min.
5. After this time, the container is removed, and the cryovials
plunged individually into liquid nitrogen (see Note 5) using
large forceps and personal protective equipment (PPE). The
vials are then transferred either into a À150
C ultra-freezer or
into liquid nitrogen storage.
6. When thawing the algae, it is important to expose the samples
to as little light as possible. To thaw, the vials are removed from
the freezer/liquid nitrogen and agitated gently, one at a time, in
a 40
C water bath until all ice crystals are gone (no longer than
2 min) (see Note 7). The thawed vial is then quickly cleaned with
70% ethanol and transferred to the laminar flow cabinet. The
contents are then transferred, by pipetting, into 20 mL of
growth media in a sterile growth flask with a vented lid.
7. This is then placed into a dark box and placed at growth
temperature. The cultures are left in the dark for 24 h. After
this time, the lid is gradually opened to allow some light
in. After 48 h of thawing, the cultures can be removed and
placed at the light intensity they are normally grown at.
8. Viability should be assessed microscopically although it is
worth noting that it can take up to 6 weeks for a strong culture
to establish.
3.4 Cryopreservation
of Macroalgae
In the Culture Collection of Algae and Protozoa (CCAP) laboratory in recent years, a range of macroalgal species have successfully
cryopreserved using slight modifications of the standard two-step
method used for microalgae (see Table 4 and Fig. 2). To successfully
cryopreserve macroalgae, the starting material must be relatively
dense, healthy, and growing well with minimal bacterial contamination. Large thalli and clumps of filamentous species should be
carefully dissected into small pieces, taking care not to introduce
contamination [14–16]. In some cases, the addition of an extracellular membrane impermeable cryoprotectant is required such as
sorbitol or polyvinylpyrrolidone (PVP).
616
Estefania Paredes et al.