This post details symbiont cell counting protocol for the 2023 Montipora capitata larval samples.

Symbiont Cell Density Counting Protocol

Based on the Putnam Lab protocol.

Materials required

• Microscrope
• Prepared symbiont cell suspension
• Hemocytometer (with included cover slips)
• P10 and P200 pipette
• DI Water and 70% ethanol (for cleaning)
• Kimwipes

Protocol

1. Clean hemocytometer and slide with 70% ethanol and DI water by squirting and wiping with a kimwipe. Place on microscope stand, turn on microscope, and visually inspect to make sure no cells are visible on grid.

2. Remove 1-2 samples from the freezer at a time and thaw.

3. Add 50 µL of DI water to the sample.

4. Sonicate sample for 10 seconds. Wipe the sonicator with a kimwipe and 70% ethanol.

5. Vortex the sample briefly right before sample is taken.

6. Take an 8 µL sample and load onto one side of the hemocytometer. Take a new aliquot from sample and fill the other side (do not fill both sides from same aliquot).

7. Using the 10X objective, count the cells on one side of the hemocytometer. Each grid is divided into nine large squares. Start by counting the cells in one of the large squares. Count enough large squares until at least 50 total cells are counted. Use a counter to help you keep track. For example, if there are 20 cells in square 1, also count cells in squares 2 and 3 to reach > 50 cells. If there are fewer than 50 cells on the whole hemocytometer, count all cells that you see in all 9 squares.

   

8. Record the number of squares you used to count and the number of cells. Count using a standardized approach - for example, only include the cells touching or on the line on the left and top sides of each square. Keep this consistent. If you count squares 1, 3, 7, and 9 on one side of the hemocytometer, count those same squares on all replicates for that same sample.

9. Repeat with other side of hemocytometer, counting the same number of squares as before. Record the number of cells.

10. Clean the hemocytometer as before with 70% ethanol and/or DI water. Get a new pipette tip.

11. Repeat above steps twice more for 6 total counts (3 hemocytometer slides with 2 sides counted per slide).

12. On all hemocytometer slides, count the same squares and the same number of squares for each biological sample.

    

13. Dispose of empty tube.

14. Repeat the process with a new sample.

Record data in notebook:

• Date
• Initials
• Sample
• Counts for all 6 replicates
• Any notes about the sample or process

References:

Schoepf et al., 2013. Coral Energy Reserves and Calcification in a High-CO2 World at Two Temperatures. PLoS ONE 8:e75049