What's In John's Freezer?

This week we conducted wallaby leg dissections for a study of the kneecaps of marsupials (pouched mammals). Placental (non-pouched) mammals like us almost all have bony kneecaps but many marsupials do not. Kneecaps do important things, acting like gears around the knee joints (e.g. this old post), and yet it is unclear why some marsupials have lost, kept or even re-evolved them as bones. So we’re investigating that and already noticed that one of our wallabies has bony kneecap(s) whereas the other doesn’t, so we’re checking out why and taking tissue samples to do histology (sectioning for microscopic imaging of tissue composition and structure) on so we can see what the knee tendon/kneecap tissues are made of. Some marsupials turn their kneecaps into fibrocartilage rather than bone or tendon and that can be impossible to identify without histology.

The wallabies are small, about 20lbs or so and just take a day or so. Like a turkey. And it’s Thanksgiving today, so here I am with a post about thawing specimens for science, rather than for food. Maybe the title will make sense now.

Stomach-Churning Rating: 7/10; thawed wallaby bits from the get-go.

Thawed lower leg and foot of wallaby. The stickers are for an old study that would take too long to explain…

This post was directly inspired by journalist Jason Bittel’s inquiry to me about my tweet on the wallaby thawing; he wondered if there might be a fun story linking thawing-for-science with thawing-for-Thanksgiving. Some highfalutin editors didn’t agree, so no printed/online story came of this, but I am not so highfalutin, hence this blog post.

Thawed wallaby forelimbs. I’m also looking into the “false thumbs” that some marsupials have (“sixth fingers”), much as elephants and other mammals may have.

Thawing is second nature for our lab’s team; we do it all the time. Avid readers will be unsurprised to learn that just about everything I’ve worked on has been frozen at some time, and thus has been thawed out at some time(s). Normally we don’t freeze if we need live tissue or undistorted tissue, e.g. to measure physiology or very fine microstructure– freezing disrupts all of that. We would instead use physiological saline solution or else a preservative like formalin. And you can only freeze and then thaw a specimen for two times or so before it becomes too useless even for anatomical study.

A small specimen like this salamander can be thawed out simply by running it under warm water for a little while or leaving it out for an hour.

We just leave specimens in a cart, or on a table or sometimes in a cold-room shelving area, for slower thawing. Space heaters tend to overdo things. We don’t do any rough calculation from some sort of thermodynamic first principles of time-to-thaw vs. specimen size (I wish we were that smart!); just seat-of-pants guessing and checking (yes, poking specimens to check their thawedness is a method of choice). Cutting things in half along the way, or skinning them, may be used to accelerate the thawing process. But it’s about as unscientific a method as we use.

The hardest specimens to thaw of course have been the largest specimens. Elephant legs can be >2 metres long and hundreds of kilograms (especially when frozen). A week at room temperature tends to work OK for getting them to a dissectable state. One has to balance the outer deterioration with the inner frigidness. We’re not so concerned about microbe growth in most cases, as one would be with a thawing turkey, and not at all about consumption. We just want to be able to dissect it and make observations, mostly via eyeballing the specimens as we dissect them,

Left hindfoot of an Asian elephant. Still frozen; this was bandthawed- I mean bandsawed- to see its internal anatomy nice and clearly. You may see this specimen again somewhere else– stay tuned! 🙂

Moisture and fluids can be a challenge: generally the rooms we thaw in are low humidity so moisture may not be an issue once the ice melts away, and we have drains nearby. We try to remove ice first or have towels to wipe/soak fluids up as thawing progresses. But if a specimen is sitting in a cart or storage bag with too much ice early on, that can thaw first and then turn the specimen into a nasty slurry of the stuff you’re interested in and the less desirable muck. So we try to avoid that.

De-thawing too early is bad. The smell gets progressively worse– and once the interior of the specimen is thawed enough, then bacteria get in there and the interior becomes a brewing ground for heat production (rather than remaining a cooler region), which accelerates decay, so we don’t want that. We have to check on thawing specimens regularly and move them to cooler storage areas, or begin dissection earlier, if the decay process is noticeably getting excessive.

Any insulation affects thawing time- so scales, feathers, thick skin, shells, fat (for a short while until it decays), and other layers will slow thawing—and may keep heat inside, if there begins to be thawing of the core. So sometimes you open up a specimen that seems dry and clean on the outside and the inside is unpleasant. But with experience that is not hard to avoid.

Thawed wallaby patella prepared for histology.

The foulest specimen I’ve thawed by far was a monitor lizard… it was shipped to me in California from Arizona when I was a PhD student. This was in August’s heat and the box of the big lizard sat thawing at the post office for 2 weeks before they contacted me and asked why a smelly box was bleeding. I came and got it and brought it back to our department but the smell was so bad it set off our building health & safety person’s alarm bells (sorry, David!) and they emailed around a “toxic alert” warning, until I bashfully made it clear that my lizard was the cause, not some toxic chemical. I got in some trouble and was very ashamed. But we put the specimen into a big tank of brine solution and the smell was reduced—the specimen may well still be preserved there 20 years later; I do wonder! Anyway, that experience was so horrendous – and I have a strong stomach—that I regularly recall it and seek to avoid a repeat. It was the most disgusting thing I’ve ever experienced. I do not recommend it.

What we tend to want to get from thawed specimens is: (1) descriptive anatomy (what connects where), and maybe (2) quantitative measurements (laborious metrics of “muscle architecture”– how much does each muscle weigh, how long is it, etc; over and over again for many muscles…). These data not only serve to tell us what makes animals different (and how this evolved) but also the data are used to test questions such as how animals work. In the case of things like wallabies, ultimately we’d love to know what their kneecaps do if they are bony or not; what difference does it make and why might there be differences? We’d spotted one wallaby already that seemed to have a bony kneecap on one leg, and a non-bony one on the other leg, so that asymmetry got us excited.

What’s surprising to learn about thawing animals for science? Well, my first thought is that it’s beautiful. I don’t tend to think of it as gross. I’ve rhapsodized about this before. Animals are wonderful inside and out, and I regularly pause during a dissection to marvel at how amazing the anatomical specializations of animals are. Simple details- shapes, colours, configurations- can be gorgeous. (Often the blood is minimal, drained out early, so that doesn’t detract from or hide the detailed imagery) The gentle yet complex path of a tendon around a joint can yield profound visual enchantment in its elegance. This is all the more true once one ponders how these complex structures evolved, and how much diversity of form and function is out there to study—and how little we know about it! We still don’t know well how to fix many problems humans have with their anatomy, and that’s orders of magnitude worst for most animals, because we don’t understand how anatomy works, or even what the anatomy is like in some cases. So that keeps me busy discovering things. Every specimen is different with surprising little variations, or big ones—sometimes there is one muscle, sometimes it is clearly divided into two muscles, in the same species or even the left vs. right legs. I love seeing those intricacies and wondering about them.

Thawed wallaby shank sliced open to show lovely digital flexors and gastrocnemius muscles. So many questions are raised by this!

If you’re thawing for Thanksgiving, or thawing for science, or thawing out family relations during a gathering, or thawing yourself out from the winter’s cold– my best wishes to you! May we all enjoy what we thaw.