Coral tissue removal, homogenization and host/symbiont separation

Airbrush tissue off frozen coral fragments, homogenize the slurry, aliquot it for physiology assays, and split it into host and Symbiodiniaceae fractions.

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

At a glance

Input	Snap-frozen coral fragment (−80 °C)
Output	Homogenized tissue slurry (“blastate”) in PBS, aliquoted as holobiont, host and symbiont fractions for downstream assays
Time	~10–15 min per fragment to airbrush and homogenize; ~1 h for the separation in Part C (estimates)
Tested on	Porites astreoides, Astrangia poculata, Galaxea fascicularis

Tissue is blown off the skeleton with an airbrush, homogenized, and split into aliquots. Part B gives the quick split used for the Porites physiology panel (protein, carbohydrate, lipid, chlorophyll, cell counts). Part C is a more thorough host/symbiont separation with washed symbiont pellets, used for enzyme assays and fraction-specific DNA.

WarningNot for RNA

Airbrushed homogenates work for DNA and physiology, but RNA from them came out degraded. For RNA, clip tissue straight from −80 °C into a bead tube instead (see DNA/RNA co-extraction).

Materials

Equipment

• Airbrush (Iwata Eclipse or similar) and a compressed air source
• Handheld rotor homogenizer (e.g. Pro Scientific Bio-Gen PRO200)
• Quart-size zip-top bags, forceps
• 15 mL Falcon tubes, 1.5 mL tubes
• Refrigerated microcentrifuge (4 °C), vortex, ice bucket
• Hemocytometer (cell-integrity check in Part C)

Reagents

• Ice-cold 1× PBS, pH 7.4 (filtered seawater also works, as in the Astrangia samples)
• 10% bleach, DI water, isopropanol wipes or 70% ethanol (cleaning between samples)
• For Part C: lysis buffer and wash buffer (below), 0.5 mm glass beads

Solutions to prepare (Part C only)

Solution	Recipe	Storage
Lysis buffer (25 mM Tris pH 8, 1 mM EDTA, 10% glycerol)	250 mL: 6.25 mL 1 M Tris pH 8 + 0.5 mL 0.5 M EDTA + 25 mL glycerol + 218.25 mL DI water	4 °C
Wash buffer	250 mL lysis buffer + 25 µL Triton X-100	4 °C

Procedure

A. Airbrush and homogenize

1. Label a zip-top bag and a 15 mL tube per sample. Pre-chill the PBS on ice.
2. Take the fragment out of the −80 °C and put it in the bag with forceps.
3. Airbrush with ice-cold PBS until all tissue is removed, including deep tissue between corallites. Use PBS sparingly: aim to keep the total volume under ~15 mL so it fits in one tube.
4. Pour the slurry into the 15 mL tube and rinse the bag with a little PBS into the same tube.
5. Record the total volume. You need it to normalize every downstream assay. Keep on ice.
6. Homogenize for 30 s on ice. If the slurry is split across tubes, homogenize each and pour back and forth to mix.
7. Clean forceps and homogenizer between samples: 10% bleach (30 s) → DI water (30 s) → DI water (30 s) → isopropanol wipe.
8. Keep the skeleton (soak in 10% bleach for 24 h, dry and label) if you need surface area for normalization.

Airbrushing video

B. Aliquot for the physiology panel

Label six 1.5 mL tubes per sample: A-Host, A-Sym-1, A-Sym-2, B-Host, B-Sym, C.

9. Add 1 mL homogenate to each of A-Sym-1, B-Sym and C.
   • C = holobiont fraction (protein, carbohydrate, lipid). Set aside on ice.
10. Spin A-Sym-1 and B-Sym at 13,000 × g, 3 min.
11. From A-Sym-1: move the supernatant (~1 mL) to A-Host (host protein, carbohydrate, lipid). Resuspend the pellet in 1 mL 1× PBS, vortex and pipette until fully dispersed. Move 200 µL to A-Sym-2 (symbiont cell counts) and keep 800 µL in A-Sym-1 (symbiont protein, carbohydrate, lipid).
12. From B-Sym: move the supernatant to B-Host (spare host). Leave the pellet in B-Sym (chlorophyll).
13. Freeze all tubes at −80 °C. Freeze the leftover homogenate (~5 mL) for ash-free dry weight.

Aliquot scheme used for the Porites July Bleaching physiology panel

C. Thorough host/symbiont separation (enzyme assays, fraction DNA)

All steps on ice or at 4 °C. Starting material: a 500 µL frozen homogenate aliquot.

14. Thaw on ice and add 0.6 mL ice-cold lysis buffer. Vortex.
15. Check cell integrity on a hemocytometer (symbiont cells should be intact).
16. Spin 6,000 × g, 2 min, 4 °C. Move 800 µL of supernatant to a new tube (host fraction). Keep the pellet (symbiont fraction) on ice.
17. Host: spin 16,000 × g, 20 min, 4 °C to remove debris. Move the supernatant to a new tube, vortex, aliquot (e.g. 600 µL DNA, 100 µL citrate synthase, 50 µL protein) and store at −80 °C.
18. Symbiont: resuspend the pellet in 200 µL lysis buffer, vortex and spin 700 × g, 5 min, 4 °C. Discard the supernatant.
19. Resuspend in 1 mL ice-cold wash buffer, spin again (700 × g, 5 min) and discard the supernatant. Repeat the wash for 5 washes total.
20. Resuspend in 300 µL lysis buffer, add 200 µL 0.5 mm glass beads, and vortex 3 min to break the symbiont cells.
21. Take the supernatant and aliquot (e.g. 200 µL DNA, 50 µL citrate synthase, 50 µL protein). Store at −80 °C.

Troubleshooting

Problem	Likely cause	Fix
Slurry over 15 mL, too dilute for assays	Too much PBS while airbrushing	Use short bursts, and work the airbrush close to the surface
Symbiont pellet contaminated with host tissue	Too few washes	Do all 5 washes in Part C, and check a drop on the hemocytometer
Low signal in lipid assays	Homogenate too dilute (600 µL input was below detection in 2021-06-22)	Use 1 mL input, keep slurry volumes low, or concentrate by vacuum centrifugation

References

• Putnam Lab coral tissue removal protocol

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Merged the airbrushing protocol (2021) and the homogenate separation protocol (2019) into one page
2021-06	–	Six-tube aliquot scheme for the Porites July Bleaching physiology panel
2019-05	–	Host/symbiont separation with washed symbiont pellets (Astrangia nutrition)