#Isolation and Culturing of Zooxanthellae from Corals ##Method from the Master’s thesis of: Ben Berman

###Purpose: The objective of this protocol is to isolate symbiotic zooxanthellae algae from corals and be able to successfully maintain them in culture. This will reveal if these algal endosymbionts can survive and grow outside their coral host, and if so for how long. Isolated algae samples will be either in F/2 or YBCII culture medium. Algae cell viability will be determined by analyzing the population of cells alive in each culture medium.

Once a baseline of isolated zooxanthellae cell viability is established, future work with isolated zooxanthellae cultures will incorporate into the growth mediums either cellulose nanofibrils (CNF) or cellulose nanocrystals (CNC). CNC and CNF are nanomaterials of cellulose that are to be tested as an artificial coral mucus. Zooxanthellae demonstrating viability in these materials will allow further testing for the therapeutic delivery potential of these Zooxanthellae integrated in an artificial coral mucus.

Below includes the isolation steps, culturing guidelines for survival, and maintenance recommendations.

***Solutions:***

• Filtered Artificial Sea Water (FASW)
• Caclium-free Artificial Sea Water (CASW)
• Double-strength Guillard’s F2 Marine Enrichment Culture Solution Medium (F2)

*Refer to previous protocols in the lab notebook to see how to prepare solutions.

###Methods: **Organism Sampling:**

• Coral samples were acquired by fragmenting laboratory established colonies of Stylophora pistillata corals from the Leon H. Charney School of Marine Sciences in Haifa, Israel.

**Algae Isolation:**

• Adult colonies were fragmented into pieces around 1-1.5 cm in length.
• Each fragment was washed with 10 mL of filtered ASW, then washed with 10 mL of Ca-free ASW (CFSW), and transferred into a fresh 10 mL of filtered CFSW with 5 mM Ethylenediaminetetraacetic acid (EDTA) for 5 min.
• Disassociation was performed by gently scraping and peeling the coral tissue from the skeleton with 10 μl sterile plastic tip at room temperature for around 10–15 min.
• The crude homogenate was passed through a 100 μm filter to remove coral debris into a 50 mL Falcon tube.
• This solution will be then washed by centrifugation 3 times at 1400 g for 20 mins, where algae will pellet on bottom, and resuspended in fresh FASW.
• A sample was analyzed under the microscope to identify cells compared to contaminants, and fluorescent microscopy was used to identify the presence of live cells using chlorophyll natural fluorescence.

**Zooxanthellae Culturing:**

• Prepare 40 mL culture flasks with either 20 mL of double-strength Guillard’s F2 Marine Enrichment Solution or 20 mL of FASW as a control.
• Gently add 200 uL of stock algae solution to the vessel while swirling.
• Close the flask and place in an incubator at 26°C with a 12/12 hour light/dark cycle and shake at 80 rpm.

**Culture Analysis:**

• The 40 mL culture flasks were first observed at a glance.
• A 1 mL sample was taken from each culture flask and put into a 1.5 mL Eppendorf tube, and examined at eye level.
• A 20 uL sample from each 1 mL sample was pipetted onto a microscope slide and examined using microscopy.
• The remaining sample in the 1.5 mL Eppendorf tube was centrifuged twice at 1400 g for 20 mins, examining after each centrifugation.
• After the 2 centrifugations, the supernatant was removed by pipetting, and the remaining pellet was submerged in 20 uL of FASW, disturbed gently by tapping the bottom of the tube to resuspend the pellet in solution, and a 20 uL sample was taking to analyze using a hemocytometer using brightfield and fluorescent microscopy.

Cultures were analyzed every 24 hours until no live cells were consistently observed in the sample.

• Then to assess the viability of the entire culture, the remainder of it was added to a 50 mL Falcon tube.
• The empty 40 mL culture flask was then washed and shaken with 25 mL of FASW to obtain reminent material, and this was poured into the Falcon tube the original culture was poured into.
• These Falcon tube samples were entrifuged twice at 1400 g for 20 mins, inspecting for a pellet after each centrifugation.
• After removing the second supernatant, the pellet was resuspended in the small backwash of the supernatant that would not be emptied from the tube by pouring, and this solution was analyzed on a hemocytometer in the same manner previously to assess for the presebce of live cells in the culture.

###Findings A total of 2 stock cultures were isolated and monitored daily using brightfield and fluorescent microscopy. Amount of available stock limited experimental replicates.

Culture 1 (least concentrated)

• Algae cells taken from top (diluted) of first algae stock solution.
• 4 replicate cultures (200 uL algae, 20 mL F2) were produced, with one 20 mL F2 culture as negative control.
• No cells observed in 24 hour sample.

Culture 2 (medium concentrated)

• Survived

Culture 3 (most concentrated)

• Survived for longest

**Conclusions**

• More concentrated cultures survived longer.
• Algae in F2 survived better compared to FASW.