Coral histology

Goal: create and execute histology protocol

Adapted from protocol by Dr. Beth Lenz

Materials and Equipment

Protocol Steps

1) Select and record plug ID of corals to be fixed

Randomly choose a sample from each tank, and record the plug ID of the sample. Note how large the coral is – this will determine what type of vessel (falcon tube or container) it will be put in for fixation.

2) Prepare and label falcon tubes and containers.

Label should include plug ID, tank number, timepoint, species (AST = Astrangia), initials, and date.

3) Calculate total volume based on number of samples and types of vessels you need to fix with.

40 mL per sample for falcon tubes; 400 mL per sample for containers

4) Filter seawater for fixative solution

Choose a 0.2 μm filter. Set up the vaccum filter housing: water will be poured into the top, vaccummed through the filter, and deposited in the bottom container.

Attach the hosing of the vaccum pump to the bottom container of the vaccum filter housing. Once hosing is secure, turn pump on and check dials to make sure there’s enough pressure.

When bottom container is full (~1 L), turn off the pump, wait for pressure to get to 0, and slowly remove hosing from filter housing. Pour the now-filtered seawater into a glass bottle. Repeat until enough is made for fixative.

5) Dilute Z-fix with filtered seawater (FSW) to a 1:4 of Z-fix:FSW

Put FSW water in hood. Get Z-fix from cabinet, put spigot on Z-fix container, and dispense enough needed for fixative. Pour desired amounts of FSW and Z-fix into labelled plastic jug (use funnel if necessary).

Zfix (10% aqueous buffered zinc formalin) is very toxic!! Use full PPE while working with it. Make sure to have two people to take Z-fix container out of cabinet, as it is bulky and heavy.

Read Product Information and Safety Data Sheet for Z-fix before use.

6) Prepare pre-labelled tubes and containers prior to sampling. Pour diluted Z-fix into individual falcon tubes and containers.

For falcon tubes, add between 30-40 mL of fixative per tube. For containers, add 350-450 mL of fixative per container.

7) If transport is necessary, secure now-filled tubes and containers in a cooler with a formalin pad in it.
8) Collect and fix samples.

Samples should be taken from tanks, removed from their individual plugs, and placed in specific tube/container. Carefully place sample in tube/container so the fixative does not splash.

If sample is not fully immersed in buffer, add more buffer until sample is fully immersed.

9) Store samples in a cool, dark place (currently in fridge for flammables)

Samples can remain in Zfix for up to 6 months

10) Pour off Zfix and replace with ethanol

Pour Zfix from sample containers into a designated waste container (once waste container is full, fill out this form to request hazardous waste pick-up). Refill the containers with the same volume of 70% ethanol.

10) Decalcify coral

In a fume good, make a 10% hydrochloric acid solution in a plastic beaker/container and place coral in solution. The coral may bubble or fizz slightly. Leave the coral to decalcify - this may take several hours or days.

To see expanded decalcification protocol, go here.

11) Once decalcification is complete, cut out 1-3 polyps and put them into individual, pre-labelled 1.5 mL tubes.

Once the coral is fully decalcified, pour off any excess HCl and put the tissue on a plastic petri dish. Using a scalpel, dissect out 3 individual polyps. Put them into the 1.5mL tubes and add 20% Z-fix to the tube. Close the tube and parafilm the top. Put the rest of the decalcified tissue in a pre-labelled 15 mL tube, and store all tubes in fridge.

If the coral polyps are very small, dissect polyps under the dissecting microscope. To see the dissecting microscope protocol, go here.

12) Ship tissue to histology laboratory.

For the GSO Astrangia project, we used Pacific Pathology for our histology needs. We requested serial section slides in H+E (Hemotoxylin and eosin stain).

If shipping 1.5 mL tubes: To prep samples for shipping, make sure the tops of 1.5 mL falcon tubes w/ samples are parafilmed. Put the freezer box with the 1.5 mL tubes in a ziplock bag and stuff it with paper towels and/or kim wipes. Close the ziplock and double and/or triple bag it using ziplock bags.

If shipping 15 mL tubes: To prep samples for shipping, make sure the tops of 15 mL falcon tubes w/ samples are parafilmed. Wrap a kim wipe around the falcon tube, place it in small vacuum bag, and seal the bag with a heat sealer. Put all bagged samples in a 1-gallon ziploc bag with paper towels; don’t overfill the bag, make sure the bag can still be closed.

Print out sheet of info about the request and URI contacts for PP and include it in the box. To ship, put the ziploc bag(s) and the printed info sheet in a box, stuff it with paper towels, and tape shut. Find the weight and dimensions of the box (will be needed for shipping). Using the UPS/FedEx website, print out a shipping label and tape it on the box; ask Hollie or other lab member how to create shipping label. Once the box is ready, put it on the shelf in the downstairs loading dock reserved for outgoing deliveries.

Pacific Pathology contact information: Pacific Pathology Inc., 9292 Chesapeake Drive, Suite 100, San Diego, CA 92123; (858) 576-9630; research@pacificpathology.com

Slide scanner at Brown:

Written on February 25, 2021