LARGE SQUID FIXING AND PRESERVATION NOTES

By Steve O'Shea
Auckland University of Technology
New Zealand
June 10, 2003

Introduction

The following is a basic step-by-step rundown of squid-handling procedures (from receiving the specimen to preserving it) that I have found effective for fixing giant cephalopods, particularly Architeuthis.

A large waist-height bench with raised lip, bench drainage, in a room with floor drainage, good lighting and ventilation are prerequisites in making the work environment a pleasant one.

Initial treatment history

Today most large squid are sourced from commercial fisheries (as incidental bycatch), and have been blast frozen at sea before being delivered to the laboratory. Stranded specimens are also almost always frozen as a means of temporary storage. Therefore most large specimens will need to be defrosted prior to examination and fixation. This is not a simple matter of removing them from a freezer and placing them onto a bench and waiting. Cephalopods weighing in excess of 70 kg (e.g., Taningia, Architeuthis, Mesonychoteuthis, Haliphron) can take several days to defrost this way, depending on ambient room temperature, during which time the more delicate parts of the animal, such as the tentacles, arm tips, suckers and sucker rings, fins and skin deteriorate, while the mantle may still be partially frozen. For best results accelerated defrosting of large cephalopods is recommended.

Defrosting

At 15—20°C a 150—200kg squid (mature female Architeuthis) can take 72 hours to defrost if simply left on a bench, but the time can be reduced to 48 hours if the squid is bathed constantly in a gentle shower of cold water, and ~ 36 hours if a running hose is also placed inside the mantle cavity. The faster a specimen can be defrosted, examined and fixed, the better its final preserved condition will be.

There are a few basic rules to follow when defrosting large squid/cephalopods at an accelerated rate.

Never use warm water.
All temptation to prise the specimen apart, especially in an attempt to disentangle the mantle from the head, arms and tentacles to accelerate defrosting, must be resisted. Walk away and let the shower of water do its trick. Giant squid (Architeuthis), in particular, are remarkably delicate animals, especially when partially defrosted, and the slightest wrench can snap an arm, tentacle, pull series of suckers from the arms or sucker rings from the suckers, and rupture the skin (already minimal in trawl-caught specimens).
When handling the mantle, great care must be taken not to exert too much pressure with your fingers, as hand and finger impressions remain that prove almost impossible to smooth over (remaining on the final fixed specimen).
Unsightly asymmetrical bulges in the mantle usually mean fish bycatch is contained within (having slipped inside during the trawl or processing). So as not to destroy the integrity of the specimen, a small (~ 5 cm) exploratory incision should be made through the mantle over the hump, and a gently running hose inserted into the incision. Should the bulge be no more than a deposit of ice then it will melt and the bulge will vanish; should it remain then expand the incision to accommodate your hand, feel around inside and extract any alien object (e.g., fish).

Only when the animal is near-completely defrosted will you be in a position to appraise its condition. You must then determine what you want to do with the specimen. If its condition is good then you should consider displaying it, in which case I have a few further recommendations:

Under no circumstances should you cut the mantle open along the ventral mid-line. Although this will expose the viscera and provide unrestricted access to alimentary and reproductive systems (in particular the stomach, digestive caecum, and reproductive organs), doing so will immediately destroy the specimen's structural integrity and aesthetic value.
A similar amount of scientific information can be discerned from the specimen by making several strategic incisions through the ventral surface of the mantle. The digestive caecum can be located and removed through a cut of ~ 20 cm length, and a smaller incision (or core sample) can be made in the posterior-ventral portion of the mantle through which ovarian tissue can be removed. The male penis will protrude freely through the mantle or funnel, thus sexing and determining his reproductive status will pose no challenge, nor necessitate a ventral mantle incision. The female nidamental glands can be examined by a mid-ventral incision of ~ 30 cm length about one quarter the distance up from the anterior mantle margin.
The oesophagus and intestine of large squid are almost invariably empty, so extensive dissection to examine their contents is unnecessarily destructive (however, the crop of large octopods is worthy of investigation (requiring a greater degree of dissection)).

It is a personal conviction that only damaged specimens of well-known giant squid and octopus species should be dissected. Given the number of museums desperate to receive specimens of giant cephalopods (of any species), retaining the integrity of the specimen should be a major consideration when preparing one of these animals for exhibit.

Precautionary or pre-fixation measures

A simple test to determine whether squid tissue is ammoniacal involves cutting ~ 1 cm3 cubes from the animal, placing them into a narrow-necked flask (test tube or other such glass container), adding several crystals of caustic potash (potassium hydroxide) and then heating the container. A quick sniff will tell you whether any ammonia vapour is present.

The pH of formalin solutions fluctuates widely when fixing ammoniacal squid, more so than non-ammoniacal species. If ammoniacal:

1. Remove representative sucker rings and hooks from the arms and tentacle clubs prior to fixing them. The sucker rings of Architeuthis have a white carbonate deposit that is invariably lost during fixing with formalin, regardless of how well buffered the solution is; these can only be preserved in buffered ethanol or isopropyl alcohol.

2. Remove the statoliths (two clean cuts).

3. Remove a piece of arm tissue, score it well, and place into a jar of 90% ETOH (for DNA analysis). Only when this tissue sample has been removed should you proceed to step 4.

4. Inject the squid tissues with 10% bicarbonate-buffered formalin solution using a 150 cc syringe (or greater), with long screw-on needle (~ 150 mm length). This introduces formalin into the tissues faster than it would otherwise penetrate the specimen if simply immersed in formalin solution. Should resistance be met then care must be taken to not exert too much pressure on the syringe plunger: unsightly clots of formalin solution can form in the tissues; if the needle is blocked then the plunger can break, resulting in a high-velocity wash of formalin solution being shot back towards your eyes; and if the needle is of the push-on variety, it has a habit of magically dissociating itself from the syringe, resulting in yet another high-velocity wash of formalin being shot everywhere. Into each of the mantle, head and arms slowly inject the following volumes of 10% carbonate buffered formalin solution:

Mantle (dorsal): ~ 2 litres injected mid-dorsally, deep into the mantle, to reach the digestive gland; about 1 litre injected in the posterior-most portion of the mantle (~ mid-fin length) to reach either testis or ovarian tissues. Mantle (ventral): ~ 1 litre either side of the ventral mid-line, in the posterior third of the mantle to reach renal and cardiac tissue; and ~ 1 litre on the animals left hand side in the posterior quarter of the mantle to reach the digestive caecum.
Head: dorsal and ventral mantle and head injections are required; ~ 1 litre of formalin solution needs to be injected into the cranium to fix oesophageal, nervous and buccal tissue.
Arms: at intervals of ~ 30 cm from the arm base, down each arm, ~ 300 ml of formalin solution, progressively decreasing the volume as the tips attenuate.

5. If the tentacles are attached to the squid carcass (an exceedingly rare occurrence), sever them at their bases and fix them separately in a 5% carbonate-buffered formalin solution in an appropriately sized bin (usually they can be coiled within a 0.75 m x 0.5 m length/width and 0.5 m depth fixing bin). They require no injection with formalin solution. They are fixed separately for three reasons:

They defrost faster than the parent carcass and need to be fixed as soon as possible to prevent further deterioration.
The palm of the club is brilliant white, the outer surface dark red, so leaving them in an oxidised formalin solution (containing the large squid) will taint their colour.
The tentacles are less buoyant than the mantle and head tissues. As a consequence they sink to the bottom of the larger fixing tank (accommodating the entire squid). The floor of the tank is where any surplus sodium bicarbonate will precipitate, and any tissues that come into contact with this will corrode. They also become difficult to locate by the end of the week in the discoloured, oxidised formalin solution, and can be overlooked when monitoring both of the formalin solution pH and overall condition of the squid.

Fixing tank and formalin solution preparation

1. Tanks that I have found best for fixing large squid have been constructed from stainless steel of dimensions 2—3 m length, 1—1.5 m width and 0.75 m depth, with close-fitting stainless steel lid.

2. For a 200 kg squid a 4:1 formalin solution/squid ratio is recommended. Pre-mark levels inside on the tank wall to indicate volume; add concentrated formalin and then salt water to make a stock solution of 10%, agitate, take a pH measurement and buffer accordingly. Formalin respirator masks are essential; as are long gloves with elastic ends (that extend to at least the elbows).

3. Sodium bicarbonate (baking soda) is my buffer of choice, as opposed to using sodium borate (borax), calcium carbonate (shell) or marble chip. Borax tends to render the tissues translucent, whereas shell and marble chip are ineffective for rapid buffering during the first week of fixation. To fix a 200 kg Architeuthis you can expect to use 10—20 kg of sodium bicarbonate.

You are now ready to add the squid to the fixing tank.

Transporting thawed specimens

Giant cephalopods are quite feeble in construction, prone to disintegrating if inappropriately handled, and have no convenient purchase points. They require support from below in order to move them.

A stretcher made up of two poles and a doubled-over, thick plastic tarpaulin is ideal for transporting squid (a canvas tarpaulin is too abrasive on squid skin). Slide the specimen onto the tarpaulin mantle-first, then tie up the tarpaulin ends to prevent it from sliding off the stretcher during relocation (this also prevents formalin leaking from the squid and miscellaneous squid slime from dribbling down your legs... and other places).

For a squid weighing 150—200 kg, recruit at least six people to assist with transportation. Should the specimen start to slide off the stretcher do not grab the head or mantle to stop the motion. The sliding momentum is sufficient to break the head from the mantle and rupture the digestive gland; either way the structural integrity or aesthetic quality of the specimen will be lost. Simply lower the stretcher to the ground and reposition the squid.

Once over the fixing tank, the two leading pole-holders need to step into the tank; this requires wearing tall gumboots or waders. The two anterior poles are then lowered and the squid is allowed to slide into the formalin bath in a controlled fashion (as opposed to tipping it in from the side or 'trying' to gently lower and release the animal from the side of the tank both of which will result in a considerable uncontrolled splash of formalin solution everywhere, in addition to possibly damaging the specimen).

Slightly inflated wine-cask bladders can be gently inserted inside the mantle (with care taken not to damage the gills) to ensure unrestricted formalin solution reaches inner tissues. These should be inserted inside the mantle only when the animal has been immersed in formalin solution.

Monitoring the squid and pH

The formalin solution in which the squid is immersed needs to be regularly monitored for the first 7 days, but particularly the first 72 hours. Although the squid is immersed in neutral formalin solution, the pH can dive to 3 or 4 if left unchecked within the first 5 hours; if left unchecked for 24 hours all suckers rings are likely to be completely destroyed. Sodium bicarbonate must be added to the solution, either in powder or slurry form, and the entire solution then manually agitated. Measure pH after the solution has been thoroughly agitated.

For best results, the formalin/squid solution needs to be monitored every 3 hours for the first 24 hours, then every 6 hours for the next 24 hours, followed by 12 hourly monitoring on the 3rd day; after the 3rd day the specimen need only be checked once a day. Once pH stabilises you need to check it less frequently.

When adding bicarbonate (to excess) to the formalin solution, care must be taken not to let it settle on cephalopod tissue. Should precipitation occur, especially on the tentacle clubs and within the club suckers, the tissues corrode. By gently gripping the anterior margin of the mantle, raising and lowering it, fresh formalin solution can be circulated throughout the mantle; this exercise should be repeated during every pH check (every 3 hours for the first 24 hours). Similarly, mucous deposits around the buccal membrane and base of arms need to be wiped free of the squid, and the arm crown periodically agitated to ensure fresh formalin solution is exposed to the inside of the arms.

By the end of the first week, the buffered, discoloured, oxidised squid and formalin soup is a revolting yellow to red-brown. Monitoring of pH is best achieved with a digital meter, as litmus paper and coloured solutions no longer give interpretable results.

For best results (in the event the specimen is destined for display), the discoloured formalin solution should be replaced with fresh solution so as not to unduly discolour the squid; if not then no damage is done to it by leaving it in. However, the specimen must remain in the formalin solution for at least a month in order for it to fix thoroughly.

Preservation

Following the month in formalin solution the specimen needs to be thoroughly soaked and the mantle cavity gently flushed with low velocity water to remove residual formalin and miscellaneous grunge. Three water changes are recommended. Thereafter the specimen can be transferred to preservative, one of either 40% isopropyl alcohol (my preference) or 70% ethanol.

Steve O'Shea 6/10/03