An undentified species of polychaete was collected on solidified methane ice in the Gulf of Mexico using the Johnson Sea Linksubmersible. Live and fixed organisms were returned to the Penn State campus a short time later.
The protocol was a conventional fixation with the exception that the fixatives were diluted in sea water from the site and the primary fixation was done aboard the research vessel from which the Alvinwas launched.
- Fix with 1.5% glutaraldehyde, 2.5% paraformaldehyde in sea water and stored at 4 C for transport to PSU
- Rinse with simulated sea water buffer (3x5min)
- Postfix with 1% osmium tetroxide in 0.1M sodium phosphate buffer for 1 hr.
- Rinse with simulated sea water buffer (3x5min) and transfer to large specimen processing containers to prepare for dehydration and critical point drying.
- Dehydrate through a gradient series of ethanol (25%, 50%, 70%, 85%, 95% and 100% (x3) for 5 minutes at room Temperature
- Critical Point Dry the samples with bone-dry liquid carbon dioxide (4 x 3 minute exchanges + 8 minute vent)
- Mount the coverslips onto an aluminum stub with a double sticky tab and colloidal silver
- Sputter-coat 10 nm of gold/palladium (2 min at 10 mAmps)
- Desbruyeres, Daniel and Andre Toulmond (1998) A new species of hesionid worm, Hesiocaeca methanicola sp. Nov (Polychaeta:Hesionidae), living in ice-like methane hydrates in the deep Gulf of Mexico, Cah. Biol. Mar. 39:93-98