Evaluating existing methods to assess the viability of Breviolum sp. in culture and development of new methodology.

Coral reefs are the most spectacular and diverse ecosystems of tropical oceans. Corals rely on the mutualistic relationship between scleractinian corals and photosynthetic dinoflagellates of the family Symbiodiniaceae (zooxanthellae) which are crucial to the health of the host cnidarian. To better understand zooxanthellae, laboratory culture model systems are one valuable tool allowing for experimental manipulation. However, the maintenance of algal cultures is expensive and labor-intensive. Cryopreservation offers a solution for long-term storage of cultures, but a system is needed to reliably assess cell viability after thawing. Multiple methods to assess the viability of zooxanthellae exist, but many of these are tested on dead zooxanthellae killed with heat which is of little use when trying to assess the viability of cells killed by freezing. Therefore, we evaluated the utility of existing viability methods to assess live vs frozen-killed Breviolum sp. (formerly Clade B) isolated from the sea anemone Aiptasia spp. Of 38 dyes tested on zooxanthellae maintained in culture, none could distinguish live from frozen-dead cells despite replicating exactly published methods or varying concentrations and staining times. For cultured cells, live vs frozen-dead cells could easily be differentiated by a shift in autofluorescence from red to bright green for the latter. In contrast, freshly isolated live vs frozen-dead zooxanthellae could be differentiated using Acridine Orange and DAPI. These could be a promising method for evaluating the viability of cryopreserved zooxanthellae.