Homogenize the coral sample with a mechanical sawtooth homogenizer for one minute until the sample is fully homogenized and no clumps are visible. For normalization, collect a one-milliliter aliquot from the homogenized tissue for Symbiodiniaceae cell counts, coral tissue protein content analysis, and chlorophyll-A estimation. If separating the host material and algal cells, centrifuge the remaining coral homogenate at 2, 500 G for five minutes at four degrees Celsius.
Transfer the supernatant containing the host material to a new 15-milliliter tube. Add two milliliters of cold, ultrapure water to the algal pellet and vortex both host material and algal pellet for two minutes to resuspend. Centrifuge all samples again and transfer the supernatant containing the host material to a new 15-milliliter tube.
After discarding the supernatant from the algal pellet, retain the pellet in the original 15-milliliter tube. Breeze either the holobiont homogenate or the separated host in Symbiodiniaceae fractions at minus 80 degrees Celsius for at least two hours. Then, lyophilize the samples overnight with a 0.01-millibar vacuum at minus 85 degrees Celsius.
After drying, weigh each sample on a laboratory balance into separate two-milliliter, plasticizer-free microcentrifuge tubes. Microscopic visualization revealed no Symbiodiniaceae cells in host tissue samples after three wash steps. Similarly, minimal host tissue was found in symbiont fractions.
However, the holobiont homogenate indicated that intracellular Symbiodiniaceae had not been released from their symbiosomes through simple airbrushing.
