Coral fixation and decalcification for TEM

Fix whole coral fragments in zinc formalin, decalcify in HCl, and post-fix single polyps in glutaraldehyde for transmission electron microscopy, keeping the remaining polyps for symbiont counts.

Version 1.0 · last updated 2026-10-05 · Used in 1 notebook entries ↓

At a glance

Input	Live coral fragments (on tiles)
Output	One glutaraldehyde-fixed, decalcified polyp per fragment for TEM, plus a homogenate of the remaining polyps for symbiont density
Time	Fixation ≥ 24 h (up to 6 months) → decalcification hours to days → polyp dissection
Tested on	Galaxea fascicularis
Imaging	Processed and imaged by the University of Miami Miami Project to Cure Paralysis TEM Core

WarningSafety

Z-fix (10% aqueous buffered zinc formalin) is toxic. Work in a fume hood with full PPE (goggles, lab coat, gloves, closed shoes). The stock container is heavy, so move it with two people. Read the Z-fix product information and SDS first. When diluting acid, always add acid to water.

Materials

• Z-fix concentrate (Anatech); glutaraldehyde (provided by the TEM core, store at 4 °C)
• Concentrated HCl; DI water
• 0.2 µm filtered seawater (FSW): vacuum filter unit + pump
• 15 mL and 50 mL tubes, specimen containers, glass bottles, funnel
• Labeled plastic jugs/bins for formalin and acid waste
• Ruler or scale bar, camera; scalpel, Petri dish; parafilm

Solutions to prepare

Solution	Recipe	Notes
0.2 µm FSW	Vacuum-filter seawater into a glass bottle	~1 L per filter run
Diluted Z-fix	1 part Z-fix : 4 parts FSW	Mix in a labeled jug in the fume hood. TODO: the original labels say “25% zinc formalin”, which would be 1:3. Confirm the ratio.
10% HCl	900 mL DI water, then add 100 mL HCl	Acid into water, never the reverse

Procedure

A. Plan and label

1. Randomly choose the fragments to fix (e.g. one per tank) and record their tile IDs.
2. Label tubes or containers with tile ID, tank, treatment, species, initials, date and the chemical (“Z-fix in FSW”).
3. Volumes: 30–40 mL fixative per 50 mL tube, or 350–450 mL per specimen container. Pre-fill before sampling.

B. Fix

4. Photograph each fragment in seawater with a scale bar.
5. Remove the coral from the tile and lower it gently into the fixative without splashing. Top up until it is fully covered.
6. Store cool and dark (flammables fridge) for at least 24 h. Samples can stay in Z-fix for up to 6 months.

C. Decalcify

7. Pour the Z-fix into the formalin waste container.
8. Refill with the same volume of 10% HCl. Leave the cap off (CO₂ is released).
9. Replace the HCl every 24 h until the skeleton is completely dissolved (hours to days, depending on size).

D. Dissect and post-fix

10. Pour off the HCl, put the tissue in a plastic Petri dish, and photograph it with a scale bar. Note which polyp you take.
11. With a scalpel, cut out one polyp and put it in a 15 mL tube with 2 mL glutaraldehyde, fully submerged. Cap, parafilm and store at 4 °C until delivery to the TEM core.
12. Count the remaining polyps, put them in a 15 mL tube with 5 mL FSW, homogenize, and store at 4 °C for symbiont density.

Troubleshooting

Problem	Likely cause	Fix
Fragment not fully covered	Container too small or fragment too large	Add fixative until covered, or use a specimen container
Decalcification very slow	HCl spent	Replace the acid every 24 h; larger fragments take days
Tissue too fragile to dissect	Over-long acid exposure	Check daily and stop as soon as the skeleton is gone

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Rewritten from the 2023 notebook protocol
2023-01	–	Protocol developed for G. fascicularis with the UM TEM core