Why Biologists Stopped Using Formalin on Deep-Sea Jellyfish

Deep within the labyrinthine archives of the world’s oldest natural history museums, millions of glass jars sit in climate-controlled darkness. Inside these vessels, suspended in pale, noxious amber fluids, are the biological records of our oceans. Most of the fish, crustaceans, and cephalopods look remarkably as they did the day they were hauled from the water—their scales intact, their carapaces rigid, their eyes locked in a permanent, milky stare.

But step into the invertebrate zoology aisles, specifically the sections dedicated to pelagic cnidarians and ctenophores, and the visual record falls apart. Inside these jars, you will not find the ethereal, pulsing, bioluminescent predators of the abyss. Instead, you will find cloudy water and a chaotic settling of brittle, yellowish debris. They look like shredded tissue paper left out in the rain, or perhaps melted plastic bags.

For over a century, the standard protocol for preserving marine life relied heavily on a single, toxic chemical: formaldehyde, diluted into a water-based solution known as formalin. It was cheap, it was highly effective at halting decay, and it was globally available.

Yet, when applied to gelatinous zooplankton—creatures whose very existence relies on a delicate matrix of water, salt, and fragile proteins—this chemical was an imperfect savior. It prevented rot, but it fundamentally betrayed the organism’s morphology. Worse, as biological science transitioned from a discipline of visual classification to one of molecular sequencing, researchers uncovered a hidden catastrophe within those glass jars. The chemical had systematically shredded the genetic code of the specimens.

The story of why biologists ultimately had to abandon the use of formalin on deep-sea jellyfish is a tale of chemical warfare at the cellular level, the rise of genomic science, and a frantic search for alternative ways to capture the most ephemeral lifeforms on Earth.

The Anatomy of a Phantom

To understand the chemical violence of fixation, one must first understand what a deep-sea jellyfish actually is.

Unlike a crab, shielded by chitin, or a fish, stabilized by a calcified skeleton, a jellyfish is an exercise in biological minimalism. Whether it is a true jellyfish (Scyphozoa), a hydrozoan, a complex colonial siphonophore, or a comb jelly (Ctenophora), the organism is almost entirely water—often upwards of 95 to 98 percent.

This water is trapped within a layer called the mesoglea, a non-living, jelly-like substance sandwiched between two exceedingly thin layers of cells: the outer epidermis and the inner gastrodermis. The mesoglea is given its structure by a highly hydrated extracellular matrix, supported by an incredibly sparse network of collagen fibers and structural proteins.

In the abyss, where pressures can exceed hundreds of atmospheres and temperatures hover just above freezing, this architecture is an evolutionary triumph. It allows the animal to reach massive sizes without the metabolic cost of maintaining dense tissue, granting them neutral buoyancy and the ability to drift silently in search of prey.

But bring that organism to the surface, and its structural integrity immediately faces an existential crisis. The moment a deep-sea jellyfish is removed from the supportive, high-pressure, freezing environment of the deep ocean, its proteins begin to degrade. The animal essentially begins to melt.

For early oceanographers hauling up nets from the deep, the immediate instinct was to arrest this decay by submerging the animal in a potent fixative. Formalin was the undisputed king of the wet lab.

The Mechanics of a Chemical Straightjacket

Formaldehyde ($CH_2O$) was first synthesized in the mid-19th century, but it wasn't until the 1890s that its tissue-preserving properties were fully recognized. When formaldehyde gas is dissolved in water at roughly 37 to 40 percent by weight, it becomes formalin. Marine biologists typically dilute this further with seawater to create a 4 to 10 percent formalin solution for preserving specimens.

When a biological specimen is dropped into this bath, a specific and aggressive chemical reaction takes place. Formaldehyde is a highly reactive electrophile. It immediately seeks out the nitrogen atoms present in the amino acids of the animal's proteins.

Once it finds these nitrogen atoms, it forms a methylene bridge ($CH_2$), physically linking one protein chain to another. This process, known as cross-linking, spreads rapidly throughout the tissue. It binds the proteins into a rigid, insoluble three-dimensional mesh. The enzymes that normally cause tissue to decompose (autolysis) are deactivated because their own structures are locked down. The bacteria that would cause rot are instantly killed.

For a muscular tuna or a heavily armored deep-sea isopod, this cross-linking preserves the animal perfectly. The muscles stiffen, the internal organs are locked in place, and the specimen can sit on a shelf for a century without decaying.

But a deep-sea jellyfish does not have dense muscle or solid organs. It has a vast expanse of saltwater held together by a fragile web of collagen. When formalin enters the mesoglea, the cross-linking is traumatic. The chemical reaction violently alters the osmotic balance. The delicate epithelial layers contract wildly, causing the animal’s umbrella-like bell to invert, crumple, or tear. The tentacles, often lined with thousands of microscopic stinging cells called nematocysts, shrink and shear off.

"The relatively large volume of mesoglea leads to distortion of the delicate morphology and poor sample integrity in specimens prepared with standard aldehyde... techniques," notes a recent methodological review by researchers attempting to find better preservation methods.

To counter this, 20th-century taxonomists developed elaborate, painstakingly slow protocols. They learned to first anesthetize the jellyfish using magnesium chloride, preventing the animal from violently contracting its muscles when it hit the toxic bath. They learned to suspend the jellies in 2 percent agarose gel before adding the formalin, creating a physical scaffolding so the animal wouldn't collapse under its own weight as the chemicals hardened it.

Yet, even with these precautions, the long-term results were bleak. Over decades, the formalin would often oxidize into formic acid, dropping the pH of the jar and slowly dissolving whatever fragile structures remained.

For a long time, this was simply accepted as the cost of doing business in marine biology. A warped, brittle specimen was better than no specimen at all. But as the 20th century bled into the 21st, the true cost of this chemical straightjacket was finally revealed.

The Molecular Shredder

The turning point arrived with the genomic revolution. By the late 1990s and early 2000s, taxonomy was no longer purely about counting the tentacles on a hydrozoan or measuring the bell diameter of a scyphozoan. The future of marine biology lay in the DNA.

Researchers realized that the deep ocean was hiding massive amounts of cryptic diversity—species that looked physically identical but were genetically completely distinct. To map the evolutionary tree of life, scientists needed to sequence the DNA of these creatures. Naturally, they turned to the millions of specimens already sitting in museum archives.

They opened the old jars, carefully snipped off pieces of preserved tissue, and ran them through standard DNA extraction protocols.

The results were catastrophic.

When researchers attempted to amplify genes from old samples using Polymerase Chain Reaction (PCR), the reactions failed. The DNA was simply not there, or more accurately, it was damaged beyond recognition.

"If a specimen is also to be used in DNA analyses, then tissues for DNA analyses must be subsampled prior to fixation in formalin because formalin rapidly degrades DNA making it unsuitable for most molecular studies," museum curation guidelines now strictly warn.

Formalin doesn't just cross-link proteins; it also cross-links DNA to surrounding proteins, and even bonds different strands of DNA to each other. This creates a tangled, impenetrable molecular knot. When biologists used enzymes like Proteinase K to try and digest the tissue and free the DNA, the cross-links held firm.

Furthermore, over time, the acidic environment of older, unbuffered formalin solutions aggressively attacks the phosphodiester bonds that hold the DNA backbone together. It causes widespread fragmentation. A healthy strand of DNA might contain hundreds of thousands of continuous base pairs. After a few years in formalin, that same strand is chopped into tiny, useless fragments, often less than 200 base pairs long.

Attempting to read the genome of a formalin deep-sea jellyfish specimen was like trying to read a classic novel that had been run through a paper shredder, soaked in acid, and glued into a solid block.

"While formalin-fixation is generally viewed as a hindrance to analysing historical genomes," researchers at the CSIRO noted, the resulting DNA is highly fragmented and the extraction process is notoriously difficult. Modern techniques can sometimes coax partial mitochondrial genes (like COI or 16S rRNA) out of relatively young formalin-fixed specimens using specialized chemical baths to break the cross-links, but whole-genome sequencing was effectively off the table.

Marine biologists faced a hard truth. Every time they dropped a newly discovered deep-sea jellyfish into a jar of formalin, they were actively destroying its genetic code. They were erasing the very data they needed most.

The Ethanol Paradox

The obvious solution was to abandon formalin and switch to the molecular biologist’s favorite preservative: high-proof ethanol.

Ethanol (ethyl alcohol) does not cross-link proteins. Instead, it preserves tissue by rapid dehydration. It aggressively pulls water out of the cells, forcing the proteins to precipitate out of solution and coagulate. Because it doesn't form chemical bonds with the tissue, the DNA remains completely intact and easily accessible for decades.

For terrestrial insects, bird tissues, or fish muscle, dropping a sample into 95 percent ethanol is the gold standard for genomic preservation. But applying this method to gelatinous zooplankton triggers a horrifying physical reaction.

Because a deep-sea jelly is nearly entirely water, the ethanol rapidly draws that water out of the mesoglea. The result is instant, total desiccation.

If you drop a magnificent, eight-inch-wide Periphylla periphylla (helmet jellyfish) into a jar of 95 percent ethanol, within minutes it will violently contract, turning opaque and shrinking down to a hard, unrecognizable wad of rubbery tissue the size of a golf ball. The delicate radial canals disappear. The gonads become indistinguishable. The tentacles fuse together.

Ethanol preserves the genetic code flawlessly, but it completely obliterates the physical morphology.

This created a severe logistical paradox for deep-sea taxonomists. To formally describe a new species of jellyfish, scientific convention requires a "holotype"—a physical specimen deposited in a museum that perfectly represents the morphology of the species. But to prove it is a new species, one must also provide its DNA sequence.

"Jellyfish specimens are usually fixed in formalin in order to preserve their morphological characters," explained Dr. Claire Rowe, Collection Manager of Marine Invertebrates at the Australian Museum, during a recent deep-sea voyage on the RV Investigator. "This preservation method makes DNA extraction more challenging compared to fresh tissues. Voyages such as the recent one to the Coral Sea allow us to take tissue samples from the jellyfish before they are fixed in formalin".

The 3 A.M. Triage: Modern Shipboard Science

This realization fundamentally changed how deep-sea biology is conducted on research vessels today. The preservation of gelatinous zooplankton is no longer a passive act of tossing a specimen into a jar; it is a high-speed, high-stakes surgical operation.

Imagine the scene aboard a modern oceanographic vessel, rolling in the swells of the California Current. It is 3:00 A.M., and the heavy winch is screaming as it hauls a MOCNESS (Multiple Opening and Closing Net Environmental Sensing System) up from a depth of 1,000 meters.

The moment the cod ends (the collection buckets at the bottom of the nets) are brought on deck, they must be immediately transferred to chilled water to prevent the deep-sea biomass from rotting. The wet lab becomes a blur of calculated, frantic activity.

Biologists carefully pour the samples into sorting trays, searching for the fragile, transparent bodies of ctenophores, siphonophores, and trachymedusae among the dense soup of krill and copepods.

When a rare deep-sea jellyfish is found, the researcher must make an immediate, irreversible decision. They cannot preserve the whole animal in formalin, or they lose the DNA. They cannot preserve the whole animal in ethanol, or they lose the body.

Instead, a delicate biopsy is performed. Working under a microscope on a swaying ship, the biologist uses micro-scissors to snip off a tiny piece of non-vital tissue—perhaps a single tentacle or a small section of the bell margin.

This tiny tissue fragment is immediately plunged into a vial of 95 percent ethanol, or a specialized molecular preservative like RNAlater, or dropped directly into a dewar of liquid nitrogen at -196°C to instantly freeze the RNA and epigenome.

The remainder of the mutilated animal is then carefully relaxed with magnesium chloride and slowly preserved in a 4 percent formalin solution to save whatever is left of its physical shape for the museum shelf.

On major survey voyages, such as the demersal surveys of the Faroe Bank, standard operating procedures dictate exact splitting. Specific specimens of leptomedusae and siphonophores are carefully hand-picked, photographed extensively while still alive, and then their tissues are divided between liquid nitrogen for biobanking, 99 percent ethanol for genomic sequencing, and formalin for morphological archiving.

It is a necessary compromise, but an ugly one. The holotype sitting on the museum shelf is forever missing the pieces that were amputated for the geneticists.

The Search for the Holy Grail of Fixatives

The frustration of this dual-preservation requirement drove marine chemists and biologists to search for a "holy grail"—a single fluid that could preserve both the fragile, watery morphology of a deep-sea jelly and its delicate, vital DNA.

In the early 2010s, a solution known as DESS (Dimethyl sulfoxide, Disodium EDTA, and Saturated Sodium chloride) gained traction. DESS relies on extremely high salt concentrations to preserve tissue without cross-linking proteins, while the EDTA binds enzymes that would otherwise chew up the DNA.

For nematodes and some robust marine invertebrates, DESS worked beautifully. But for highly gelatinous deep-sea species, the extreme salinity caused severe osmotic shock. The jellies still shriveled, albeit slightly less violently than in pure ethanol. As researchers noted in a study comparing fixatives, DESS was "adequate for DNA analyses but led to distortion of morphological characters".

Then came more unconventional experiments. In 2021, a team of biologists studying the development of ctenophores (Mnemiopsis leidyi) published a highly unusual finding. They had struggled for years to preserve the delicate, watery morphology of comb jellies for advanced laboratory techniques like in situ hybridization (mapping where specific genes are expressed within the intact animal). Aldehydes ruined the samples, and alcohols collapsed them.

Out of desperation, the researchers turned to the automotive aisle of a hardware store. They discovered that adding Rain-X®—a commercial, hydrophobic glass treatment used to repel water from car windshields—radically improved the morphological preservation of gelatinous zooplankton.

The active ingredients in Rain-X, which include siloxanes and synthetic polymers, interacted with the highly hydrated extracellular matrix in a way that supported the mesoglea without violently dehydrating it. By using specific ratios of this commercial glass treatment, researchers were suddenly able to fix delicate ctenophores ranging from microscopic juveniles to adults larger than 3 centimeters, keeping their intricate comb rows and delicate epithelial layers perfectly intact for weeks.

While it sounds absurd to treat deep-sea biology with a windshield wiper fluid, it highlights the sheer desperation of the field. The unique physical properties of deep-sea jellies demand solutions that break all the traditional rules of specimen curation.

Digital Jars and Laser Light

Despite these chemical innovations, a growing faction of deep-sea biologists began to argue that the entire premise of "collecting" and "fixing" a specimen was inherently flawed.

When you drag a delicate, gelatinous predator up from 2,000 meters deep inside a trawl net, the physical damage is already done long before the animal reaches the formalin bath. The abrasive mesh of the net shears off tentacles. The crushing weight of other captured animals bruises the bell. The sudden change in temperature and pressure induces massive physiological shock.

For scientists like Dr. Steve Haddock of the Monterey Bay Aquarium Research Institute (MBARI), the solution was not to build a better jar. The solution was to leave the animal exactly where it was.

With the advent of high-definition camera systems mounted on Remotely Operated Vehicles (ROVs) like MBARI’s Tiburon or the Schmidt Ocean Institute’s SuBastian, the definition of a biological "specimen" underwent a profound evolution.

Consider the discovery of Crossota millsae, a breathtakingly beautiful species of deep-sea jellyfish found off the coasts of California and Hawaii at depths below 1,000 meters. The species is visually stunning, featuring a translucent bell, vibrant yellow and red internal pigmentation, and a fringe of fine tentacles.

When researchers first encountered C. millsae, they did not just blind-trawl it. They observed it in situ using ROVs. Because they were able to film the animal alive and unbothered in its natural environment, they discovered something astonishing: this bathypelagic species was brooding its young. The female medusae had tiny, fully formed juvenile jellyfish developing inside her subumbrellar space.

If those specimens had been caught in a standard MOCNESS tow, the violent turbulence of the net would have stripped the brooding juveniles away. If the animal had been dumped into formalin, the subsequent crumpling of the bell would have obscured the reproductive structures.

Instead, the ROV’s video footage captured the true morphology, behavior, and ecology of the animal. The high-definition 4K video essentially became the holotype.

Today, in situ imaging is the primary mode of morphological preservation. Lasers are projected from the ROVs across the animal’s body to provide precise, millimeter-accurate measurements of its bell diameter and tentacle length while it swims freely. Advanced systems like DeepPIV (Particle Image Velocimetry) shine lasers through the water to reveal exactly how the jelly pumps water and catches prey.

Only after the animal has been thoroughly documented digitally, mapped in 3D, and observed in its ecological context does the ROV gently capture it using specialized, soft-sided suction samplers.

Once brought to the surface, the physical specimen is almost entirely surrendered to the geneticists. With the morphology already preserved perfectly on hard drives, the scientists no longer need to rely on toxic vats of formaldehyde to save the shape. The animal can be rapidly frozen in liquid nitrogen to preserve not just its DNA, but its delicate RNA and epigenetic markers, giving researchers a complete map of its evolutionary history.

The Archive of the Future

If you walk through the basement of a major natural history museum today, you will still see the endless rows of jars filled with formalin. They remain a vital historical archive, a chemical snapshot of the oceans from an era before genetic sequencing and robotic submarines.

There is even a quiet revolution happening within these old collections. Advancements in bioinformatics and specialized genomic protocols are finally allowing researchers to bypass the devastating cross-linking caused by formaldehyde. By using modified Micrococcal nuclease assays and advanced whole-genome sequencing techniques, geneticists are beginning to crack open the shattered DNA inside these historical specimens. They are figuring out how to read the shredded book.

But for newly discovered species, the era of the chemical straightjacket is over. The toxic, eye-watering smell of formaldehyde is slowly disappearing from the wet labs of oceanographic research vessels.

The realization that we cannot force the deep ocean into a glass jar has fundamentally elevated the science of marine biology. By accepting the ephemeral nature of these gelatinous creatures, scientists have learned to meet them on their own terms.

Today, a "preserved" deep-sea jellyfish is no longer a cloudy, brittle lump sinking to the bottom of a toxic bath. It is a dual entity. It exists as a flawlessly clean string of digital code—A, T, C, and G—stored on a genomic server, detailing its lineage back millions of years. And it exists as a brilliant, high-definition video file, showing the creature pulsing silently through the crushing blackness of the abyss, perfectly intact, alive, and glowing in the dark.