Ink and labels

Labelling specimens may seem pointless when you have a couple of skulls on a shelf, but it becomes absolutely essential when you start to have specimens all over the place. And while you might think that the choice of the pen you use to write down each specimen ID doesn't matter, it actually does. I recently realised that the ink on the labels on some of my specimens was starting to fade, probably due to light exposure. Turns out commercial pen should be avoided, and that "archival pens" (using pigment based ink instead of dye based ink) are much more suited for that purpose. So you might want to consider this when labelling your specimens, especially since light exposure is not the only thing that might threaten your precious identification system!

Label of a kitefin shark (Dalatias licha) jaw where the ink has faded due to light exposure.

This and other similar issues are covered in a very interesting poster by Amy Davidson, Samantha Alderson & Marilyn fox that can be found here.

Rattlesnake

I'm currently working on a large (c. 1.3 m) timber rattlesnake (Crotalus horridus) for a veterinarian. The specimen was in great shape, and I didn't notice anything special when I dissected and skinned it prior to cleaning. However, once cleaned I was very surprised by the bony growth present on numerous vertebrae, sometimes completely locking them together. I still have to do some research, but from what I read and discussed with a couple of people, they might be some kind of cancerous lesions. The skull also exhibits minor deformations.

Multiple views of various vertebral segments. Only the most serious deformities are pictured here. Scale bar=1 cm.

Several views of the articulated vertebral column.

The right dentary also exhibits slight deformation.

Hedgehog take two

Remember the hedgehog skeleton I posted a few months ago? Well, it was badly damaged by careless students soon after. This was partly my fault because I articulated with as little wire as possible (all carefully hidden) and no additional support, for a free standing finished mount despite the knowledge that it would be used in zoology practicums and it wasn't meant to be a display only specimen.
That being said, it's also due to the incredible carelessness of some students when handling natural history specimens. The need to handle those properly and carefully, especially when it comes to teaching collections which are used by generation of students, cannot be stressed enough.
Well, lesson learned, and I spent some time repairing it and reinforcing it with wires and additional support. Unfortunately, a toe was lost in the process.

I also added a brass plaque to the base, which always adds to the finished piece I think.

 Right side view

 Detail of the scapular belt area

 Detail of the right leg


Despite the fact that I don't like at all to come back on a specimen I consider finished, this was an interesting experience, since I usually don't build skeletons with that much wiring although I kind of like the look of it.

It's now back in the university teaching collections, let's hope it stays in one piece this time!

Smooth-hound shark skeleton

Remember the common smooth-hound shark of my first post? Well, I recently finished it, so here are a few pictures of the finished specimen, and of the cleaning process as well.

 Common smooth-hound (Mustelus mustelus) skeleton.

And now a bunch of WIP pictures:

Tail region during the initial dissection. The fins were eventually detached for the body, but it is important to know their precise location along the vertebral axis first.

Head region, the same careful dissection is performed before separating the following "units": skull and jaws/gills (this is the tricky part)/dorsal fin/scapulocoracoid/pectoral fins.

Cleaned parts in ethanol prior to drying. Despite the small size of the specimen, I still took the skeleton apart in more manageable pieces to monitor carefully the drying process.

Sclerotic capsules, dorsal fin, tail, scapulocoracoid, neurocranium and part of the vertebral column during the drying process.

Gills ready for drying. This was the most difficult part to work with, and I could not get them fully intact.

Assembly of the skeleton, here a ventral view of the neurocranium and gills.

Above view. The scleral capsules have been added. A carefully bent brass rod is used as attachment and support for the scapulocoracoid cartilage.

Scapulocoracoid cartilage and pectoral fins added.

Dorsal fin ready to be attached to the skeleton. I used brass wire of different gauges for everything, but I'm toying with the idea of incorporating clear acrylic in the next one I prepare.

Getting there...

Pelvic fin  ready to be attached. A thin brass rod is fastened on the underside of the puboischiadic bar.

Finished skeleton, waiting to be mounted on its stand.

 Display stand in progress. I wanted to avoid the usual two support rods for something more original. My original idea completely failed, but then I was suggested to recreate the outline of the shark body using the wire, and I really liked the idea, and think it turned out really nicely. I had to add an additional support rod at the front though, a single central rod was not enough, and I placed it too far back on the stand, meaning it didn't look quite right if alone.

 Various views of the finished skeleton.

Really enjoyed working on this specimen, which was a fantastic learning experience. Of course it is far from perfect, but I'm looking forward to correct those mistakes on the next specimen I do.

Clearing and staining

For the past months I've been playing around with this kind of stuff:

 Juvenile cornsnake (Pantherophis guttatus) that was cleared and stained last year. The Alcian blue excess was not completely removed by the KOH and we probably didn't use enough alizarin.

This is called clearing and staining, and a quick google search will provide you with loads of really cool pictures, and an equally large amount of protocols so you can try out this technique by yourself.
Basically, most protocols are derived from Dingerkus and Uhler (1977), although this technique is much older than that, and much details can be found in "Clearing and staining skeletons of small vertebrates", by Davis, Gore & Osgood, and which is available here.
Basically, tissues undergo an enzymatic digestion using trypsin. This is the clearing stage. Then the specimen is double stained, first using Alcian blue, which stains cartilage blue, then with Alizarin red, which stains bone a deep red colour. Specimens are then stored in a glycerin solution, which has an index of refraction very close to that of collagen, which helps "transparize" the specimen further.

In addition to looking truly beautiful, this is a very useful technique in biology, still widely in use today. It allows the study of the skeleton of very small vertebrates without the need for "classical" osteological preparations, which are especially problematic with this kind of material. It also routinely used for the study of ontogenetic patterns of ossification, along with more modern techniques such as X-ray microtomography. A third staining agent, Sudan black  B is also sometimes used as a nerve stain on previously cleared and double-stained specimens.

While on the subject, check out these specimens, cleared and stained by Adam Summers:
Some really cool fish

A friend of mine recently sent me a sample of a product called visikol, which is basically a clearing agent used for microscopy. I've yet to give it a try (which I should do very soon), but I've seen some very interesting preliminary results. It seems to clear the specimens very quickly, the tricky part being the staining process. Will probably post the results over here when I start experimenting with it.

Juvenile black scorpionfish (Scorpaena porcus) recently finished. This one took didn't clear well, probably due to the improper use of trypsin. However the stains worked quite well. Notice how the scales are deeply stained by the Alizarin red.

Close up on the jaw of a juvenile skate (Raja sp.). The tesserae are clearly visible

Reference:
Dingerkus G, Uhler L. 1977. Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technology, 52: 229-232.

Quick one

My work area with a variety of herps and fish

Busy right now, new stuff coming soon!

Small spotted catshark skulls

I've been cleaning a few small spotted catshark (Scyliorhinus canicula) skulls lately, and took some pictures to illustrate various stage of the cleaning process.

The heads prior to preparation. All of them are under 10 cm long.

Step 1: The heads are soaked in very hot (but not boiling!) water for a few hours. The water may need to be changed a couple of times as it cools and gets cloudy.

Step 2: Scraping. As the heads soak in hot water, the tissues are scraped using a scalpel and a fine pair of tweezers. 

During the scraping process, care should be taken not to disconnect the hyomandibulars, ceratohyals and basihyal if those are to be kept with the skull. On larger skulls those might be disconnected for easier handling, but on small skulls, it is easier to keep them attached. The nose area is also very delicate due to the small cartilage flaps in the nasal capsules, and to the rostrum which consist of 3 small unconnected cartilage rods in this species.

Step 3: Whitening and further scraping

Once the initial scraping is finished, the skulls are transferred to a mix of diluted H202 and ethanol. This step will whiten the specimen, and soften the remaining tissues which can be scraped further, leaving a very clean specimen. They are then rinsed thoroughly in hot water and transferred to 95% ethanol.

Step 4: Drying. 

The most common technique used to dry cartilaginous material is freeze drying. It can take some times, but restricts warping of the cartilage to a minimum. I'm still new to this technique however, and these particular specimens were dried in very strong ethanol for a couple of weeks. Note that the hyomandibulars separated from the chondrocranium on the above specimens. This will happen if the specimens are scraped and/or handled too much during this step, and they'll need to be glued back later on.

Finished specimens.

Multiple view of a finished specimen. The hyoid arch was kept in place. In order to avoid warping, small perspex sheets were used to hold the rostrum during the final air drying stage.

I'm basically using the same process, with only minor variations on all the chondrichthyan specimens I'm working on, but need to look further into the freeze drying technique in order to reduce warping.