Symbiodiniaceae cell density (hemocytometer counts)

Count symbiont cells in coral larvae or tissue homogenates on a hemocytometer, and normalize to larva number or coral surface area.

Version 1.0 · last updated 2026-10-05 · Used in 3 notebook entries ↓

At a glance

Input	A vial of larvae (20 per vial) or a resuspended symbiont fraction from an airbrushed homogenate
Output	Symbiont cells per larva, or cells per cm² of coral surface
Time	~10 min per sample for 6 replicate counts (estimate)
Tested on	Porites astreoides larvae; Galaxea fascicularis homogenates

Materials

• Compound microscope (10× objective)
• Hemocytometer with its coverslip
• Glass Pasteur pipettes
• 0.2 µm filtered seawater (FSW)
• Pestles (larvae) or a homogenizer
• Kimwipes only for cleaning (anything else scratches the hemocytometer)

Before you start

You need a normalization value for every sample:

• Larvae: the number of larvae in the vial.
• Adult tissue: the total homogenate volume (recorded when airbrushing) and the skeleton surface area.

Procedure

A. Sample preparation

Larvae (20 per vial)

1. Check the vial number against the master spreadsheet.
2. Homogenize the larvae in the vial with a pestle and 50 µL FSW.
3. Add 250 µL FSW while rinsing the pestle into the vial, for 300 µL total. Record the exact volume.
4. If you also need protein from the same larvae, move 200 µL to a new vial labeled <vial#>-P and freeze it at −80 °C (see Total protein).

Adult homogenates

5. Use the symbiont fraction resuspended in a known volume of PBS (tube A-Sym-2 in Coral tissue removal and homogenization). Vortex well before loading.

B. Load the hemocytometer

6. Put the coverslip over the center of the hemocytometer.
7. With a glass Pasteur pipette, put a small drop at the notch and let capillary action pull it under the coverslip. Load the same way every time: uneven loading is the main source of variation.
8. Place it on the microscope stage at 10×.

C. Count

9. Count all symbiont cells in the 9 large squares (the 3 × 3 grid bounded by triple lines). Cells touching the boundary lines count as inside.
   • Symbiont cells are round with no ridges, golden-brown to green, and refractile.
10. All 9 squares together are one replicate. Count the other chamber for a second replicate.
11. Mix the sample by pipetting between loads and repeat until you have 6 replicate counts.

D. Calculate

For a standard improved Neubauer hemocytometer, each large square holds 0.1 µL, so the 9 squares hold 0.9 µL. (TODO: confirm the model of hemocytometer used; the grid volume differs between models.)

\[ \text{cells mL}^{-1} = \frac{\overline{\text{count}}_{9\ \text{squares}}}{9} \times 10^{4} \]

• Per larva: cells mL⁻¹ × sample volume (mL) ÷ number of larvae
• Per area: cells mL⁻¹ × total homogenate volume (mL) ÷ surface area (cm²). Account for any dilution, e.g. if the pellet was resuspended in a different volume than it came from.

Clean-up

• Wipe the hemocytometer and coverslip with a Kimwipe only.
• Rinse pipettes with DI water between samples and before storage. Return the hemocytometer to its case.

Troubleshooting

Problem	Likely cause	Fix
Replicates vary a lot	Uneven loading or cells settling	Load the same volume the same way each time; pipette-mix before every load
Too many cells to count	Sample too concentrated	Dilute 1:2 or 1:5 in FSW and multiply back
Hard to tell symbionts from debris	Host tissue fragments	Use the washed symbiont pellet (Part C of the tissue protocol)

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Rewritten from the 2018 notebook protocol; added calculation and adult-homogenate input
2018-08	–	Original protocol for P. astreoides larvae