dinoflagellate which is commonly related with toxicity in red tides
in Galicia and elsewhere. The techniques used were a two-step
traditional cryopreservation, slow cryopreservation using passive
freezing systems, vitrification, vitrification with ultra-fast laser
warming, and encapsulation-vitrification. We tested both DMSO
and glycerol at 10% and 15% as cryoprotectants. After cryopreservation, post-thaw studies were made to observe culture viability
(recovery and density counts to compare them with controls).
A. minutum showed extreme sensitivity to cryoprotectant exposure (even stepwise). We observed that dehydration events are
lethal to this species and it did not survive any of the cryopreservation treatments. After vitrification with ultra-fast laser warming, we
have observed that the cells were intact, the membrane was not
detached, and the cell looked morphologically alive. However, cells
failed to regain motility or divide. At the Marine Biological Association in Plymouth, UK, the dinoflagellate strains PLY 701 Prorocentrum triestinum, PLY 626 Prorocentrum micans, and PLY
632 Scrippsiella trochoidea failed to cryopreserve successfully using
various methods. The benthic dinoflagellate strains, PLY
440 Amphidinium sp., PLY 450 Amphidinium carterae, and PLY
734 Amphidinium carterae, however, were cryopreserved and reanimated successfully. This is supported by similar findings for this
genus at the CCAP.
3.6 Cryopreservation,
Long-Term Storage,
and Viability
Conservation in liquid nitrogen (À196
C) will ensure the indefinite preservation of cells, as has been reported for many cell types
[e.g., 17–19]. Ultralow temperature (À150
C) freezers can also be
used for storing cryopreserved samples, but appropriate backup
systems are required in case of power failure. Once cells are stored
at these temperatures (and maintained at temperatures below the
glass transition temperature), their viability is theoretically only
affected by background radiation, which at normal levels will take
2000 years to become a hazard to stored cells [20]. The viability of
cryopreserved microalgae stored in conventional À80
C or À20
C
freezers is suboptimal and limited in time compared to storage in
liquid nitrogen [21].
When undertaking routine cryopreservation, culture collections usually qualitatively assess post-thaw viability using a combination of short-term and long-term factors. Immediately postthaw, cell motility and morphology can easily be assessed under
the microscope. Subsequently, culture recovery can be assessed by
simple visual assessment of the color of the culture flask. Quantitative methods to assess post-thaw viability include cell counting
(manual counting, flow cytometry, in vivo fluorescence) or use of
a vital stain such as DAPI.
Cryopreservation of Algae
619
in Galicia and elsewhere. The techniques used were a two-step
traditional cryopreservation, slow cryopreservation using passive
freezing systems, vitrification, vitrification with ultra-fast laser
warming, and encapsulation-vitrification. We tested both DMSO
and glycerol at 10% and 15% as cryoprotectants. After cryopreservation, post-thaw studies were made to observe culture viability
(recovery and density counts to compare them with controls).
A. minutum showed extreme sensitivity to cryoprotectant exposure (even stepwise). We observed that dehydration events are
lethal to this species and it did not survive any of the cryopreservation treatments. After vitrification with ultra-fast laser warming, we
have observed that the cells were intact, the membrane was not
detached, and the cell looked morphologically alive. However, cells
failed to regain motility or divide. At the Marine Biological Association in Plymouth, UK, the dinoflagellate strains PLY 701 Prorocentrum triestinum, PLY 626 Prorocentrum micans, and PLY
632 Scrippsiella trochoidea failed to cryopreserve successfully using
various methods. The benthic dinoflagellate strains, PLY
440 Amphidinium sp., PLY 450 Amphidinium carterae, and PLY
734 Amphidinium carterae, however, were cryopreserved and reanimated successfully. This is supported by similar findings for this
genus at the CCAP.
3.6 Cryopreservation,
Long-Term Storage,
and Viability
Conservation in liquid nitrogen (À196
C) will ensure the indefinite preservation of cells, as has been reported for many cell types
[e.g., 17–19]. Ultralow temperature (À150
C) freezers can also be
used for storing cryopreserved samples, but appropriate backup
systems are required in case of power failure. Once cells are stored
at these temperatures (and maintained at temperatures below the
glass transition temperature), their viability is theoretically only
affected by background radiation, which at normal levels will take
2000 years to become a hazard to stored cells [20]. The viability of
cryopreserved microalgae stored in conventional À80
C or À20
C
freezers is suboptimal and limited in time compared to storage in
liquid nitrogen [21].
When undertaking routine cryopreservation, culture collections usually qualitatively assess post-thaw viability using a combination of short-term and long-term factors. Immediately postthaw, cell motility and morphology can easily be assessed under
the microscope. Subsequently, culture recovery can be assessed by
simple visual assessment of the color of the culture flask. Quantitative methods to assess post-thaw viability include cell counting
(manual counting, flow cytometry, in vivo fluorescence) or use of
a vital stain such as DAPI.
Cryopreservation of Algae
619