#Calcium-free Artificial Sea Water (CASW) Protocol ##Method from the Master’s thesis of: Ben Berman

Calcium-free Artifiical Sea Water (CASW) is a contaminant free medium ideal for the isolation of Symbiodiniaceae (Zooxanthellae) from coral tissues. The absence of Calcium (Ca) reduces Ca cell adhesion allowing symbiotic alage to be dissociated from their coral hosts.

###Methods:

• Obtain a large (1 L - 2 L) Erlenmeyer flask, and place it on a stir plate with a stir bar gently placed at the bottom of the flask.
• Measure 1 L of deionized H₂O, and add the majority (750 mL - 800 mL) to the flask. Set the stiring at a gentle pace to swirl the water.
• Obtain the chemicals in Table 1, measure them to their apprpriate masses, and add them in the flask.
• Add the remaining deionized H₂O to the flask so that the final volume is 1 L, and let it mix until the contents are fully disolved.
• Remove the stir bar from the flask. In a sterilized chemical hood, pour the 1 L solution through a sterilized 0.22 uL filter into a sterilized 1 L media storage bottle.
• Seal the bottle tightly in the chemical hood, and place in storage until needed.

Table 1: Chemical ingredients and masses in 1 L of deionized H₂O in preperation of CASW medium.