This protocol is for counting Symbiodinium cells in various coral tissues. In terms of sample preparation and final counts, please be aware of what type of tissue you are using (i.e. larvae, juvenile spat, or adult tissue).

Additional data needed

• Depending on the type of tissue sample, you will need the a size or volume measurement to normalize your Symbiodinium counts.

Equipment needed

• Compound microscope
• Haemocytometer
• Glass pasteur pipette
• 0.2 micron filtered seawater
• Pestles or homogenizers

Sample preparation

a) Coral larvae (20 larvae per vial)

1. Retrieve the correct vial (always double check with the master spreadsheet).
2. Homogenize the larvae in the vial with the pestle. Add 50 μL of filtered seawater to help homogenize (keep track of this!).
3. Add 250 μL filtered seawater to the vial for a total volume of 300 μL. Use the 250 μL to rise excess homogenate from the pestle into the sample vial.
4. Subset 200 μL into a separate vial for total protein analysis.
   • Label the vial as original vial # and -P (e.g. 181-P).
   • Place in a new labelled freezer box and put it in the -80°C.

b) Coral spat (juveniles)

Loading the haemocytometer

1. Place the specialized cover slip over the middle of the haemocytometer.
2. Using the glass pasteur pipette, load the homogenate into haemocytometer through the notches on the top and bottom of the haemocytometer. Only a small amount of homogenate is needed. Place a drop onto the notch and let the capillary action draw the homogenate towards the center of the haemocytometer.
3. Loading the haemocytometer consistently is key for uniform distribution of Symbiodinium!
4. Carefully place the haemocytometer under the compound microscope under 10x magnification

Counting

1. Place the haemocytometer under the compound microscope at 10x magnification
2. You will see a grid with nine boxes (3 by 3) outlined with 3 solid lines. These are our counting grids. Within each of the nine boxes there are 12 smaller boxes.
3. Count all Symbiodinium cells within the 9 large boxes.
   • Anything that is on the 3 lines counts as inside the box (i.e. count it)
   • Symbiodinium cells should be
   • Transparent, green, and can see
   • Circular with no ridges
4. All nine boxes will count as one counting replicate. Repeat on the other side of the haemocytometer for another replicate. Complete a total of 6 replicate counts.
5. Mix homogenate with pipette between counts to ensure it is homogenized

Cleanup

1. Wipe haemocytometer and coverslip with a kim wipe. Only use a kim wipe because anything else will scratch the haemocytometer
2. Rise pipette with DI water between vials and for storage
3. Put haemocytometer back into the case.

Happy counting!