Symbiont cell density protocol

Goal: Determine symbiont cell densities in Astrangia samples from 2021 experiment

This protocol was adapted from the Putnam lab.

Materials & Equipment

  • Compound microscope
  • Symbiont cell suspension
  • Hemocytometer
  • Glass cover slip
  • Glass pipette & bulb
  • Counter
  • Vortex
  • DI water
  • Kimwipes

Samples should be aliquoted from homogenate generated post-airbrushing. See airbrushing protocol here.

Protocol

  1. Thaw sample homogenate
  2. Clean hemocytometer and slide with 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.
  3. Vortex sample to mix/homogenize.
  4. Use glass pipette to mix sample by pipetting up and down. Then take a small amount and load one side of hemocytometer (with the cover slip on it) until liquid front completely moves across the glass slide.
  5. Take a new aliquot from the sample and fill the other side of the hemocytometer (do not fill both sides from the same aliquots).
  6. Using the 10x objective, count cells in one side of the hemocytometer.
    • Each grid is divided into 9 large squares.
    • Count until you reach 100 total cells (or count all the squares). Keep track of how many squares you count.
    • Start counting in the upper left hand square and continue with the corner squares. If you have not reached 100 cells after counting the corner squares, count the other squares in the grid until you reach 100 cells.
    • Record the number of squares counted and the number of cells.
  7. Repeat with other side of hemocytometer, counting the same number of squares as before.
  8. Clean hemocytometer as before with DI water. Clean glass pipet with a tube of DI water.
  9. Repeat above steps twice more for 6 total counts.
    • Note: Each count should count the same number of the nine large squares. For example, if the first count for sample X requires 3 squares to count 100 cells, 3 squares should be counted on all subsequent counts for sample X.
  10. Make sure avg # of cells/square is consistent (calculate std dev and coefficient of variation- should be less than 15%).
  11. Multiply # of cells/square by 10^4 and the dilution factor to get cells/mL.
  12. Multiply by homogenate volume to get total cells.
  13. Normalize to surface area to get cells/surface area.
References

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

Written on January 18, 2023