skeletal articulation (fwd)

From: Pita Admininstrator (pita@whale.wheelock.edu)
Date: Thu Jan 10 2002 - 12:51:06 EST


Marine Mammal Skeletal Preparation and Articulation

Doreen Moser1, Rebecca Duerr1, and Jack Hawkes2
1The Marine Mammal Center, 1065 Ft Cronkhite Marin Headlands, Sausalito,
CA 94965; moserd@tmmc.org; pelaquin3000@yahoo.com
2 Calif. Dept. of Fish and Game, Marine Wildlife Veterinary Care and
Research Center, 1451 Shaffer Road, Santa Cruz, CA 95060;
jhawkes@ospr.dfg.ca.gov

Poster presentation at the Biennial Conference on the Biology of Marine
Mammals
November 28-December 3, 2002; Vancouver, B.C., Canada

Although there are many ways to prepare bones for scientific and
educational collections, there are few references that outline the
various methods. Here we discuss the advantages and disadvantages of
different methods in skeletal preparation and articulation. We outline
natural and chemical processes, and list contacts or supplies needed for
these processes.

Permits
In the United States, federal permits are required to obtain and prepare
any marine mammal specimen. For collection and permit guidelines,
contact the National Marine Fisheries Service for cetacean, otariid, and
phocid species; or the U.S. Fish and Wildlife Service for odobenid, sea
otter, polar bear, and sirenian species. For other countries, check
with the appropriate government agency for permit requirements.

Specimen Preparation Techniques
1. Fresh Water Maceration
Maceration is a simple method that is commonly used. Carcasses are
soaked in freshwater-filled containers, which accumulate bacteria that
aid in flesh decomposition. Choose a container that fits the entire
carcass suck that it can be completely submerged in water. Plastic
buckets with lids work best. Some workers add manure to the containers
to act as a bacterial step-up. Maceration degreases bones to some
extent, however this method has a limited ability to remove oil from
large bones, such as cetacean bones. If this oil is not removed, it may
leach out over time and result in discolored, odorous bones. Once oily
bones have been macerated to remove flesh, they may require further
degreasing (see Chemical Maceration section).

Small bones may have their flesh removed quickly, on the scale of a few
weeks, while larger bones may take many months for the flesh to
decompose away. A skull may soak for a couple of weeks to several
months. Cutting away accessible flesh prior to soaking will speed up
the process. Monitor containers periodically to keep the water level
high enough to cover the bones, and to observe the maceration progress.
Once the flesh has decomposed, bones should be removed promptly from the
maceration water to prevent them from being damaged by bacterial
action. Bones of newborn or juvenile animals must be closely monitored,
because they tend to fall apart when macerated. Scrubbing and
high-pressure hosing can assist with flesh removal. A gentle stream of
water may help clean residual tissue from delicate nasal turbinates.
After maceration, bones are usually boiled. Care must be taken to not
lose teeth and small bones when dumping maceration water or when
applying streams of water.

Pros: Natural, no chemicals, inexpensive, less time intensive
Cons: Odorous, may take several months, bones may still need to be
degreased

2. Boiling
This method involves cooking bones in boiling water until the flesh
falls off. The process works best with bones that were first
macerated. Adding one cup of Tide laundry detergent helps degrease the
bones while boiling. This method requires boiling containers of a size
appropriate to the carcass in question. In order to not confuse left
and right limb bones, it is best to remove them from the carcass and
place them in labeled bags such as stockingettes or other strong
boilable flow-through bags. Doing this also helps prevent the loss of
tiny bones amongst the debris of boiled flesh. For ease of later
assembly, one may also wish to bag other body sections such as the
sternum or sections of the vertebral column. Small bones may be boiled
in a regular dedicated cooking pot on a hot plate or other heat source
in a well-ventilated area (preferably outdoors). Use a lid to keep the
water from evaporating during boiling. Keep the water level high enough
to cover the bones. Cetacean bones may require a large container such
as a 50-gallon barrel and a powerful heat source such as a propane
burner. One set up used successfully by Lee Post of Homer Alaska (see
additional contacts) has the barrel held up on cement blocks with a
propane powered weed burner as the heat source. A metal stovepipe with
an elbow bend is used to direct the heat at the bottom of the barrel.
In some circumstances, bones too long to fit in the barrel, such as
whale jawbones, may be boiled on end and then flipped over to boil the
other end. Macerated bones should be boiled until the water no longer
has froth. Cooking times vary widely, generally in proportion to the
size of the bones. For instance a medium sized pinniped skull takes 6
to 15 hours of boiling. Once boiling is complete, bones should have any
residual flesh removed by hand and should be allowed to dry thoroughly,
ideally in the sun.

Pros: Natural, no chemicals, inexpensive, less time intensive
Cons: Odorous, bones can soften or fall apart especially in young
animals, requires very large boiling containers with a sufficient heat
source for large cetacean bones
Additional Contacts: Lee Post - (907) 235-6247, boneman@xyz.net

3. Saltwater Maceration
Flow-through systems or soaking bones in an open oceanic bay is ideal,
but is not an accessible method to all. The animal parts should be
placed in a wire cage with mesh small enough to capture the smallest
bones and staked out to a pier or other stable structure. Marine
organisms will consume the flesh and over time (six months to one year)
bones should be void of most flesh. Once the flesh has been eaten away,
bones should be dried and bleached in the sun.

Pros: Natural, no chemicals, inexpensive, less time intensive
Cons: Odorous, need to be near saltwater bay or estuary, bones can be
lost from enclosures and organisms may consume bones.
Additional Contacts: Jim Borrowman, Stubbs Island Charters - (250)
928-3185 stubbs@island.net

4. Dermestid Beetles
Dermestid beetle colonies are an efficient method to remove flesh.
Several museums with large collections use this method. Beetles ingest
flesh and bones are almost void of flesh. The colony however, must be
maintained at 70-80°F and require numerous specimens to feed them. They
can be fed dry dog food in between specimens. Colonies often require a
large space (from refrigerator to shipping container size). Beetle
colonies are ideal for smaller specimens, but it is difficult to
maintain a colony big enough for large cetacean bones.

Before feeding a specimen to a beetle colony, most of the flesh first
should be flensed off and the brain and other soft tissues removed. If
the specimen is macerated, the brain and soft tissue may be easier to
remove. Then, the specimen should be dried. The flesh should have a
'jerky-like' texture. The carcass can be placed in the colony whole or
in sections. The colony quickly consumes most of the flesh. For
instance, a pinniped skull may take two days and a porpoise skeleton may
take two weeks. Although beetles will consume the flesh, this method
does not degrease the bones.

Pros: Natural, no chemicals, less time intensive, inexpensive (after
set up costs)
Cons: Not recommended for large bones, bones still need to be
degreased, beetles must be fed when there are no specimens in
preparation, set up costs include obtaining beetles and colony
container.
Additional Contacts: Jim Thomeson, National Marine Fisheries Service
National Marine Mammal Laboratory - (206) 526-6316
California Academy of Science, Department of Ornithology and Mammology -
(415) 750-7177

5. Burying
Burying may be the only practical option for large specimens. It is
inexpensive (except in labor) and uncomplicated. Burial does have
several disadvantages. It is slow (1-10 years), and difficult to
monitor (specimens must be dug up periodically to check progress).
Small bones, such as flipper or pelvic bones, are easily lost. Burying
these bones in wire cages or mesh bags may prevent loss. Another option
is to remove the flipper bones and prepare them separately. Burial
sites must be carefully marked and recorded to prevent losing the entire
specimen. Additionally, soil bacteria and chemicals may damage bones.
Finally buried bones are quite odorous. Therefore, buried specimens may
not be suitable for indoor display.

Pros: Inexpensive, ideal for large specimens
Cons: Bones can become lost or decompose, odorous

6. Chemical Maceration
Chemical maceration works quickly, however large tanks and heaters may
represent a substantial investment. Specimens prepared by chemical
maceration often need no further degreasing or bleaching. Specimens may
be completely or partially disarticulated. Removal of specimens from
the soak with the intervertebral disks intact greatly facilitates
articulation, and produces a more realistic articulation.

Chemical holding tank: The chemical holding tank used at the California
Department of Fish and Game’s Marine Wildlife Veterinary Care and
Research Center was built around a salvaged fiberglass tank. It holds
800 liters and can hold specimens up to two meters long. Tanks can be
salvaged (a discarded bathtub, for example), or built from plywood
reinforced at the seams with fiberglass tape and coated with polyester
or epoxy resins. Note that tanks will be heavy when filled (800 liters
weigh 800 kg); they can be strengthened with 2x4 or 2x6 lumber laid on
edge. Tanks can be heated with aquaculture heaters; the tank mentioned
above uses an 1800-watt heater and thermostatic controller (Aquacenter
Inc. (800) 748-8921). Heated tanks should be insulated with one inch
polystyrene basement insulation (available from lumberyards). Tanks
containing corrosive solutions should be fitted with locking covers and
warning signs.

Terg-a-zyme, available from Alconox (914) 948-4040, is an enzymatic
detergent that can be used in a 1% solution (by weight) to strip bones.
Terg-a-zyme can be used cold or hot, and can be disposed of down the
drain. It is relatively expensive (approximately U.S. $16 for a
four-pound container), and relatively aggressive. Specimens must be
monitored closely to prevent damage, especially if used hot.

Pros: Can be used cold or hot
Cons: Relatively expensive, can damage bones quickly, especially when
used hot
Additional Contacts: Richard Baldwin, University of California at Santa
Cruz, Anthropology Department - (831) 459-5382, baldwin@cats.ucsc.edu

Potassium Hydroxide is available under the name 'caustic potash flake'
for approximately U.S. $8 for a 50-pound sack. It should be used at
0.5-1% by weight and kept at approximately 110°F. With the proper heat
source, the specimen can be left to macerate. A well-flensed specimen
can be degreased and void of flesh in three to five days. Used solution
may be neutralized with muriatic (hydrochloric) acid and disposed of
down the drain. For all chemicals, please check with local authorities
for proper disposal. Finished specimens are rinsed in water and
scrubbed in hot soapy water with a nylon bristle brush. Potassium
hydroxide slowly decomposes cartilage, therefore sternebrae,
intervetertebral disks, and flipper ends are easily preserved.
Potassium hydroxide cleans, degreases, and deodorizes bones prepared by
other methods, such as burying. Bones from an adult elephant seal
soaked in a 0.5% solution subsequent to burying in an attempt to
degrease and especially deodorize them showed minor damage after
approximately three weeks immersion and were clean enough that indoor
mounting was an option.

Pros: One step cleaning, degreasing and bleaching, quick, inexpensive
after initial investment
Cons: Requires chemical-resistant tank, heat source

7. Degreasing Agents
The following are chemicals that can be used to remove grease from
specimens, in which the flesh has been removed. These chemicals can be
used on specimens that were buried, macerated, or placed in a bug
colony.

Ammonia is an inexpensive and simple process for degreasing bones.
Bones void of flesh can be soaked in 50% ammonia or ammonia hydroxide.
Soak the specimen for two to seven days. Once you remove the specimen,
soak it in warm water to flush out the ammonia. Used ammonia can be
disposed of down the drain

Pros: Inexpensive, easy disposal, degreases bones
Cons: Caustic
Additional Contacts: Jim Thomeson, National Marine Fisheries Service
National Marine Mammal Laboratory - (206) 526-6316
California Academy of Science, Department of Ornithology and Mammology -
(415) 750-7177

Hydrogen Peroxide soak can whiten and degrease bones, but over time the
bones may flake. Use a concentration of 2-5%. Note that most
commercial hydrogen peroxide is 35% solution and should be diluted.
Simply soak bones void of flesh in hydrogen peroxide for less than one
hour. Bones soaked longer than one hour may begin to deteriorate.

Pros: Inexpensive, whitens bones, non toxic
Cons: Bones may flake over time

Specimen Articulations
1. Overview and References
When articulating a specimen, a number of decisions must be made prior
to beginning the assembly. First, will the specimen be indoors or
outdoors? If it is to be outdoors, the bones will need to be coated
with epoxy or latex paint for protection from the elements, lest the
specimen rapidly decay. Second, will the display be for research
purposes, where the bones should be minimally altered, or for
educational purposes, where sturdiness might be more important? Third,
a pose must be chosen, including whether the specimen will be supported
from the top (hanging), the bottom, or the side. It is wise to do a
number of sketches of possible poses given the circumstances of the
intended display. Look at photographs or watch live animals with the
intent of seeing how their skeletal structures move in various
positions. Observing emaciated animals can be quite instructive, if the
opportunity is available. Examining radiographs can provide valuable
information on vertebral spacing and posture, plus unravel the mysteries
of wrist and anklebone placement and spacing. If possible, take
radiographs of whole limbs before cleaning the flesh off the bones. For
drawings of pinniped wrist and anklebones and their relationships see
The Pinniped Project: Building a Sea Lion Skeleton by Lee Post. This
handbook may be obtained from the author (see contact information at
Boiling section). For drawings of seal, sea lion, manatee, and dolphin
anatomy, see drawings by Sentiel Rommel in “Gross and Microscopic
Anatomy” Chapter 9, CRC Handbook of Marine Mammal Medicine (2nd edition.
Dierauf, L. and Gulland, F. eds. CRC Press, Boca Raton, FL. 2001. 1063
pp.). For general comparative anatomy, see Kenneth V. Kardong,
Vertebrates (2nd edition, Boston, WCB/McGraw Hill, 1998). For one of
the few books on general skeletal preparations, see Milton Hildebrand,
Anatomical Preparations (Berkeley, U.C. Press, 1968).

2. Materials
Marine mammal skeletons are generally mounted with a metal rod running
from inside the back of the skull, down drilled holes through each
vertebra’s centrum, to the logical stopping point. In pinnipeds and sea
otters, this rod stops at the fused sacral vertebrae; in cetaceans and
sirenians it continues until the vertebrae are sufficiently small to
merit a change of support rod diameter. The rod may be up to half the
diameter of the smallest vertebral centrum through which it will pass.
If the spinal column is prepared whole, with vertebral disks left
intact, a rod can be covered with polyethylene tubing and run down the
neural canal.

Some workers prefer to use threaded stainless steel, with or without a
polyethylene tubing covering. Others however, find stainless steel to
be hard to bend and drill. Stainless steel can be easily cut with a
Dremel cut-off wheel (Dremel Corporation www.dremel.com). Some workers
prefer aluminum rods due to their lighter weight and ease of bending.
Others however, prefer to not use aluminum because glues do not stick to
it well. Some workers prefer galvanized, threaded rod.

A variety of adhesives can be used. Cyanoacrylate adhesives and hot
glue are often used to tack bones in place prior to a final application
of space-filling material between bones. Aesthetically speaking, a case
can be made both for and against using bone-colored space-filling
compounds. The skeleton will appear as a more homogeneous whole if
bone-colored compounds are used; however, some prefer the more dramatic
appearance of a darker material to offset the individual bones. For
structurally strong, space-filling materials the authors have used Magic
Sculpt (Tap Plastics, www.tapplastics.com) and marine epoxy (West
Systems www.westsystems.com). Both materials can be rather expensive in
large quantities, but may remain feasible within limited budgets for
small to medium sized specimens. Magic Sculpt has the advantage of
being the consistency of clay while hardening to a strong plastic
overnight. It also smoothes with water while wet and is sandable and
paintable after curing. Marine epoxy can be more difficult to work with
but is stronger overall. Unthickened, it has the consistency of honey,
but it can be thickened with microfibers to the consistency of peanut
butter. Thickened, it is roughly bone colored and can be shaped after
curing with an abrasive wheel in a Dremel tool. Other workers have used
clear or white silicone sealant built up in layers with success.
Five-minute clear epoxy may be useful in some circumstances but is runny
prior to setting and tends to yellow with time. Hot glue has been used
for a variety of purposes, but is not very strong and appears to loosen
its grip over time.

To simulate cartilage, such as the costal cartilages, many materials
have been used. Silicone applied in neat layers over heavy gauge wire
has worked well, as have various kinds of tubing. Rigid materials tend
to not be very successful cartilage replacements. To be thorough when
replacing cartilage, remember to consider the cartilaginous extensions
of the digits in otariids.

Flipper bones may be pinned together with stainless steel or aluminum
wire or small gauge threaded rod segments. Sixteen gauge wire is
adequate for small flipper bones or and for tail vertebrae. If the
flipper bones are of sufficient size, 11-gauge aluminum chain-link
fencing ties work well and allow easy bone positioning prior to the
addition of space-filling material between the bones.

Scapulas are separated from the rib cage by thick, powerful muscles;
hence, the limb may be spaced away from the ribs by short sections of
clear, stout vinyl tubing or other material. The pectoral flippers are
attached by pinning and epoxying the entire front limb together in the
desired position, then wiring the scapula to the rib cage at judicious
locations, with the tubing sections added as spacers. The pectoral limb
may also be mounted to thin, bendable plastic sheet (e.g.
polycarbonate), such that the limb arches over the rib cage without
touching it. Plastic sheets easily crack, therefore, drill any holes
carefully and do not overtighten attachments.

Missing bones may be replaced by a number of methods. If the missing
piece is extant but not to be used in the display, a mold can be made of
it and a replacement cast. Bondo, Plaster of Paris, Magic Sculpt,
silicone rubber, and urethane have been used to make one and two part
molds. Silicone rubber or urethane available from Tap Plastics is ideal
but may be expensive for large pieces. Plaster of Paris is less
expensive but may be more troublesome to work. Replacement material
should not be subject to significant shrinkage during curing and should
produce a good level of detail. TAP Quik-Cast Polyurethane Casting
Resin System (Tap Plastics) works quite well with silicone and urethane
molds. The casting material is watery when mixed, flows well into
molds, and is ivory colored when cured. This method can also serve to
make a plastic resin replacement from a hand sculpted clay or wax model
(useful when the original is missing). Keep in mind the need for air
bubbles to escape and the method by which you will remove the finished
product from the mold. Silicone molds usually require tiny air vents
cut at strategic locations. A sharpened three millimeter metal tube
works well for removing a tiny core of the mold material at air-trapping
locations. One-piece silicone or urethane molds must be judiciously
slit to allow removal of first the original and later, the replacement.
When slitting these molds, use an Exacto knife and cut in a zigzag
fashion to allow for later reapposition of the mold. A straight cut
makes for difficulty in proper alignment later.

Skulls are often stabilized and teeth attached by the careful
application of white glue. Coat the skull in a 10% white glue mixture
for a protective coating. The right and left mandibles may be attached
at the symphysis with white glue, silicone or epoxy. The mandible may
be attached to the skull by running a wire through the temporomandibular
joint by drilling through the chondylar process of the mandible and the
mandibular fossa of the temporal bone. Sixteen gauge wire curled into a
tight coil at the ends allows the wire to be tightened into the desired
position. Round needle-nosed pliers are needed for this. To mount the
mandible in an open-mouth position, wire through the joint as above but
leave enough play in the wire to allow bending to the desired position.
Temporarily brace the mouth open with any material, such as crumpled
paper. Tighten the wire as needed. Fix the jaw in position by adding
space-filling epoxy to the TM joint. Remove the temporary mouth brace
when the epoxy has cured.

A number of methods have been used to attach the skull to the spinal
rod. For small skulls, metal pins (such as 3/16 inch threaded steel)
may be mounted into the occipital condyles, through the atlas vertebra,
and into the axis. The spinal rod, in this case, may stop at the axis
or atlas vertebra. For larger skulls, the spinal rod may extend into
the skull cavity itself. The skull cavity will need to be filled with a
rigid material such as plaster of Paris or hard-setting insulation spray
foam. All perforations in the skull should be masked off before filling
the cavity, and the occipital condyles should be masked as well, to
prevent the material from adhering to the outside of the skull if it
expands beyond the condyles. Once the cavity has been filled, a
carefully positioned hole may be drilled to accommodate the rod.
Alternatively, a metal support brace or cradle may be custom built to
hold the skull in a less altering manner, this however requires welding
skills and access to equipment. This is a preferred method when working
with large specimens.

3. Pinniped and Sea Otter Notes
Pinniped and sea otter preparation techniques are very similar. A
realistic spinal posture can be obtained by arranging the vertebrae such
that the faces of adjacent vertebrae centra are parallel to each other.
The natural bend of the neck comes from the shape of the individual
cervical vertebrae, rather than from the spaces between them. Phocids
have an extremely deep “S” curve to their neck position. This means
that when a seal’s head is tucked in, the skull is quite close to the
scapulas, nearly forming a “U” shape with the spine curving to the
ventral area of the neck. This allows them the ability to quickly dart
their head forward to catch prey. Otariids also have a significant
curve to the resting neck posture. Pinnipeds and sea otters naturally
assume dynamic poses when alive, and have extremely flexible spines.
Skeletal articulations will better educate the viewer if they are
designed with naturalistic fluid postures.

California sea lions have 15 ribs on each side. The costal cartilages
do not merge. Numbers 1-9 attach to the spaces between the sternabrae.
Numbers 10-12 reach the ziphoid but are not attached; their tips lie
ventral to the ziphoid. Numbers 13-15 float completely and taper in
length. On a large animal there may only be 3-4 cm between the end of
rib number 15’s cartilage and the patella, as the rib cage is quite
large overall. Sea lions have a relatively small abdomen.

Vertebral spacing varies from one area of the spine to another. Use
radiographs or take careful measurements to maintain accurate spacing,
lest the finished skeleton be foreshortened. For example, the
California sea lion has much larger spaces between the lumbar vertebrae
immediately cranial to the pelvis than between the thoracic vertebrae;
this allows the spine to bend to rotate the hips forward for quadrapedal
walking. When they do this, the calcaneus (heels) remains very close to
the spine, and the trapezoidal shape formed by the four points of the
knees and heels pivots as a unit at the lumbar vertebrae.

4. Cetacean Notes
Cetaceans pose several problems. Because cetaceans need to maintain
neutral buoyancy their bones are filled with oil, which tends to leak
out slowly over time. Preparation by chemical maceration in potassium
hydroxide helps remove much of the oil, but bones may still need long
soaking in ammonia or other degreasers.

Because their bones are not weight bearing, they are composed almost
entirely of cancellous bone, with very thin cortices. For this reason
cetacean bones are quite vulnerable to water intrusion if mounted
outdoors, as is often desirable because of their large size. Water
intrusion invariably causes rotting, and may increase skeletal weight
dangerously. A blue whale skeleton at the Long Marine Lab in Santa
Cruz, California was sealed with a shellac-based primer and painted with
latex house paint. This system works reasonably well, but requires
periodic maintenance. For a gray whale skeletal mount, now in the
design phase, they are considering using a clear penetrating epoxy
sealer (Smith and Co., www.smithandcompany.org), a layer of marine
epoxy, and a linear polyurethane topcoat. However the sealer is
expensive (U.S. $75/gl) and requires several coats; it also necessitates
using respirators while it is being applied, as the fumes are noxious.
This sealer is used at The Marine Mammal Center for cetacean bones
displayed outdoors. It protects bones from the elements for several
years.

Finally, small cetaceans like dolphins and porpoises have large numbers
of almost-identical teeth. A great deal of time can be saved by pulling
teeth first and then keeping them in order, for example in modeling
clay.

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