them until all ice melts, avoiding swirling the sample too much
to prevent mechanical damage. Wipe each tube with 70% (v/v)
ethanol prior to further manipulation. Aseptically empty the
contents of each tube into a sterile culture flask and dilute by
adding 20 mL of fresh culture media (equivalent to the
medium used for the original culture).
8. Incubate at the same temperature as the initial culture. For the
first 24 h, it is recommended to incubate in the dark (by covering flasks with aluminum foil) to allow for a gradual reactivation of photosynthetic mechanisms. Step 7 is also advised to be
performed under low light.
9. Viability assays: reanimation success can be assessed simply by
visually checking the color of the culture and periodically
examining cells under the inverted microscope over time (typically several weeks). Cryopreserved cultures typically exhibit a
relatively long lag phase before reaching exponential growth
and may therefore take several weeks to recover normal
growth.
In order to illustrate the efficiency of this method, statistics for
employment of the method in recent years by the RCC are presented below. Since 2011, in total this method has been applied to
1765 strains (almost exclusively marine) from the RCC, with 1551
(88%) having been successfully reanimated. This high success rate
in part reflects the fact that in most cases, cryopreservation has been
applied to species/strains that are expected to be resistant. Of
successfully cryopreserved strains, the majority are green algae
(Chlorobionta) (399), cyanobacteria (332), alveolate parasites
(188), diatoms (184), haptophytes (170), and bacteria (143). Ten
dinoflagellate strains have been successfully cryopreserved. Of the
strains for which cryopreservation was not successful, the majority
are diatoms (94), green algae (Chlorobionta) (57), and dinoflagellates (13). In terms of percentage of total strains of different groups
held in the collection that have been cryopreserved, cyanobacteria,
green algae, bacteria, and alveolate parasites are all above approximately 40% (see Fig. 1). Note that in many cases, the percentage is
low because cryopreservation has not yet been attempted for many
strains in the group.
A full list of species that have successfully been cryopreserved
using the above method is given in Table 1 (note that in many cases,
there are species for which cryopreservation was successful for some
strains but not others). Diatoms can be used as a test case to
investigate the factors that may influence cryopreservation success.
In total, 278 diatom strains have been tested at the RCC,
184 (66%) of which have been successfully reanimated after cryopreservation. There is considerable variability between taxonomic
groups (genera or classes) within the diatoms in cryopreservation
610
Estefania Paredes et al.
to prevent mechanical damage. Wipe each tube with 70% (v/v)
ethanol prior to further manipulation. Aseptically empty the
contents of each tube into a sterile culture flask and dilute by
adding 20 mL of fresh culture media (equivalent to the
medium used for the original culture).
8. Incubate at the same temperature as the initial culture. For the
first 24 h, it is recommended to incubate in the dark (by covering flasks with aluminum foil) to allow for a gradual reactivation of photosynthetic mechanisms. Step 7 is also advised to be
performed under low light.
9. Viability assays: reanimation success can be assessed simply by
visually checking the color of the culture and periodically
examining cells under the inverted microscope over time (typically several weeks). Cryopreserved cultures typically exhibit a
relatively long lag phase before reaching exponential growth
and may therefore take several weeks to recover normal
growth.
In order to illustrate the efficiency of this method, statistics for
employment of the method in recent years by the RCC are presented below. Since 2011, in total this method has been applied to
1765 strains (almost exclusively marine) from the RCC, with 1551
(88%) having been successfully reanimated. This high success rate
in part reflects the fact that in most cases, cryopreservation has been
applied to species/strains that are expected to be resistant. Of
successfully cryopreserved strains, the majority are green algae
(Chlorobionta) (399), cyanobacteria (332), alveolate parasites
(188), diatoms (184), haptophytes (170), and bacteria (143). Ten
dinoflagellate strains have been successfully cryopreserved. Of the
strains for which cryopreservation was not successful, the majority
are diatoms (94), green algae (Chlorobionta) (57), and dinoflagellates (13). In terms of percentage of total strains of different groups
held in the collection that have been cryopreserved, cyanobacteria,
green algae, bacteria, and alveolate parasites are all above approximately 40% (see Fig. 1). Note that in many cases, the percentage is
low because cryopreservation has not yet been attempted for many
strains in the group.
A full list of species that have successfully been cryopreserved
using the above method is given in Table 1 (note that in many cases,
there are species for which cryopreservation was successful for some
strains but not others). Diatoms can be used as a test case to
investigate the factors that may influence cryopreservation success.
In total, 278 diatom strains have been tested at the RCC,
184 (66%) of which have been successfully reanimated after cryopreservation. There is considerable variability between taxonomic
groups (genera or classes) within the diatoms in cryopreservation
610
Estefania Paredes et al.