Clearing and Staining Fishes

Clearing and Staining Fishes


So we’re here with Dr. Caleb McMahan, who is the collection manager of Fishes here at the Field Museum, and
today we’re going to be talking about clearing and staining. What is clearing
and staining? Caleb: So, clearing and staining is a process where we take an entire
specimen and we clear that specimen down and use different stains to stain
different parts of the skeleton. Emily: So how is that process actually done — how do you
get from something that’s, like pickled in a jar like this, to something that you
can literally see through? Caleb: In general the process is that we start with a specimen, so here we have a species of
cichlid from Nicaragua and Costa Rica and so we take this cichlid and the
first thing we do — and, again, some people do things differently, but I — a lot of times will
take the eyes out. If it’s a really thick fish we might take the skin and scales
off of it. And then we’re going to take out the the guts. We take the gonads out
and we store those in a vial back in the jar so that if later someone says I
had the skeleton but I want to see what sections of the reproductive tract look
like they can still go back and do that so then we’re left with our fish and we
first dehydrate them, in really strong ethanol, like 95% ethanol, and then we start
the process. And in general the two big phases: one, we’re going to stain them so
we’re going to take the fish and we’re going to use a stain called alcian
blue. And we’re going to put the fish in this solution and it’s going to stain the
cartilage blue. And then we’re going to then use the red dye called alizarin red. Things with calcium — calcium phosphate — will be stained red by this
dye. The clearing of it to clear away the tissue used trypsin which is an
enzyme that digests proteins. It’s found commonly in in vertebrate digestive
tracts. And trypsin eats away at the at the tissue, the proteins, but it leaves
the collagen. So you can see I can still pick this fish up. So it’s still an
actual specimen. Emily: It’s like a jelly specimen! Caleb: Right, right. It’s still an actual fish
specimen.. it’s not just a pile of bones in the tray. And then we store it in
glycerin, so this — Emily: That’s what the gooey, syrup stuff…Caleb: The gooey kind of stuff. What’s really neat about storing it in glycerin
is that glycerin and collagen have the same refractive index. So, how light
passes through – so when you put this on a tray on a light box like this you just
get the skeleton. Emily: So… that’s how– that’s how we’ll get invisibility cloak technology. Caleb:
Probably coming from cleared and stained fish, yes. Emily: Yeah, that will inspire how we develop that. Why would you use this process over just something like — you know, creating an
x-ray or CT scan? It seems like it would be a little bit more evolved than that. Caleb:
we still use x-rays a lot, radiographs for looking at skeletons. But the nice
thing about cleared and stained specimens is that we have a a 3D visualization of
the skeleton, so X-rays don’t give us that. Sometimes the location of certain
cartilages or how they vary across different species or groups of species
can tell you a lot about their evolution, their development, and anatomy. And in
fishes a lot of times the larve look nothing like the adults and so you can
track how certain bones change through develop, and how the general morphology
of the fish changes and those make really interesting studies. Emily: So you can try to
essentially track the growth of an animal, or a species throughout different
stages. Caleb: Right, right. Emily: Are fish the only thing that you clear
and stain? Caleb: They’re not the only thing. So these are fish examples, but we also have
a lot of examples from other vertebrate groups as well. These are amphibians and
reptiles. So here we have a snake you can very nicely see the vertebral column wrapped
up here. There’s a frog, and the turtles are interesting because they look about
the same size but you can see the blue one, there, is– that’s a cartilaginous
skeleton, and so you’ve got an embryonic turtle there. Emily: so that was a baby turtle. Caleb: That’s a baby baby turtle. Emily: Aww Caleb: And then you have this smaller turtle — the small turtle here
with the red skeleton, that’s, ah.. Emily: it’s just a smaller — small species of turtle, but
it’s an adult. Caleb: Right. Emily: So what are some of the discoveries or novel concepts that have
been learned from using this process? Caleb: in my own research I’ve used cleared and
stained specimens to look at their teeth and morphology of their jaws, and how
they look the same or different between species, or across groups of fishes. Emily: So kind
of how something could move in relation to another thing? Caleb: Ah, or in their
appearance, but that’s that’s another really, really interesting way that
people are using cleared in stained specimens for biomechanics, and understanding —
I mean, even here we had this incredible diversity of morphology in fishes. And this particular species has this very long — it’s called an ascending process — off of
this upper jaw bone. Emily: It’s like a big extra bone up its forehead? Caleb: Right and so and what this fish does is you can see with the mouth closed but to open the mouth you can use
this specimen and you can see how the jaw opens. Emily: Oh wow! Caleb: Yeah, so using cleared and stained
specimens allows us to look at how these things move and how these things feed. Emily: So does this process destroy the
specimen at all? Because it — obviously it looks like it’s kind of invasive. Caleb: It
doesn’t destroy it — it it changes the the the form of the specimen. Instead of
looking like this, or like this, it’s now a different appearance, and you can
answer different questions with it. A lot of times I think, you know, we associate
to tell different species apart or or groups of fishes — that it’s not all just
always based on external anatomy or morphology. So, sometimes the
characteristics of their of their skeleton which tell us what species it is.
And- and so, sometimes – for instance – this is one of the type specimens of a knife
fish. And so- so- we clear that this was cleared in stained as part of the
description of the species to make sure that the skeleton — and show how the
skeleton –was different from other species it related to. Emily: Oh wow, so
scientists don’t don’t see this process is destructive at all the informative. Caleb:
Yes, absolutely. Emily: Can you clear and stain a human? Like, can you clear and stain me when I
die? Caleb: it would take a lot of tripsin. Emily: It would be very expensive. Caleb: And a lot of glyserin. Emily: But you could do it. Caleb: It would be a bit expensive Emily: Ok. I think that’s a good use of
scientific funds. …. probably not. It still has brains on it.

100 thoughts on “Clearing and Staining Fishes

  1. I might be wrong, but I'm pretty sure it should be diaphAnisation, not with an O. It's the process of making something diaphanous, nothing to do with sound (phonos).

  2. I don't know, I think it'd probably be worth doing this to a human to see what we can learn about our own bodies. Depending on how expensive it is it might be best to leave that to an eccentric billionaire instead of draining public funds, but isn't doing weird things with their corpses what eccentric billionaires are for?

  3. The stingray(-ish?) and turtles look amazing. Couple of questions:
    – Can mammals be cleared too? He only mentions "other vertebrate groups"
    – Can you reintroduce slight color variations in the soft tissue? It would be amazing to be able to distinguish muscle and such on a translucent level while still be able to see the bones

  4. This is one of the most fascinating processes! I'm so glad you did a video about it. Here's Adam Summers of the University of Washington talking more about it https://www.youtube.com/watch?v=haopSRCuPdo

  5. Yet another amazingly interesting behind-the-scenes episode, Emily ^^ Diaphonization… another word I had never heard before. Seems to be pretty useful. And it looks pretty cool, too.

  6. Sooo…storage of these specimens after they've been cleared and stained is just in one of those partially filled clear containers? Cuz it seems like they'd slosh around a bit, potentially becoming damaged :/

  7. nice video,,, please, I need to talk with a enlgish native to improve my speaking skills, i will have a international test to study in USA. and I nedd to talk with someone, Please, add me to kmy Skype count agonzalesdiaz

  8. I'm glad Emily asked the question I was wondering the whole way through the video… could you clear and stain a human? XD

  9. Man, i would love to have a job like Emily! She gets to dip into all types of sectors of science (and look at some of the coolest things on earth) and spread knowledge that a lot of people would never get to see!

  10. if you dont like the word "fishes" i'd have to ask if you prefer "childer" to "children", or "eyen" to "eyes", or "thing" to "things"… if "fishes" is incorrect then surely all the others are too? ;p

  11. I've gotta try that as a pick up line. "That would take a lot of trypsin, ladies." Thank you for the awesome episode! you guys always rock.

  12. How come only the gonads are kept? Are reproductive systems in general much more interesting than the intestines?

  13. I like the stain technique, sure, but what really hits me is Emily. She has a couple titles.
    Chief Curiosity Correspondent on the obligatory twitter: https://goo.gl/5GP5pQ
    Science communicator, as described on Wiki: https://goo.gl/THTgrB
    Some A's for those of you who have Q's: https://youtu.be/VxQkIJFDEDo
    Allow me to be candid. I'm experiencing the biological drive to combine DNA molecules with her.

  14. I want a coffee table with "Cleared and Stained" specimens as decorative pieces inlaid into it an lit from below!
    Give me one now! I'll pay extra for the "Brainscoop" logo is somewhere on the piece.

  15. that is one of the coolest things i have ever seen. not only is it a fascinating process, it also looks very aesthetically pleasing for some reason.
    Is this something a layman could do as well, or are these chemicals restricted in their availability outside of professional scientific research? Also, could you encase these specimens in something like clear urethane resin or would that not work due to their gelatinous surface?

  16. Awesome process! I found some of these teeny tiny stained specimens in 2 inch jars for sale when I visited Tokyo! I regret not buying one of them now 🙁

  17. Fantastic! This is great example of the vast riches found on YouTubes video servers – the channel "for the people". Many thanks for your video. I just had to chime in and say, "COOL!".

  18. I've made a few calls and haven't had any luck–do funeral homes use some sort of trade name for diaphonization, or am I just pronouncing it wrong?

  19. VERY COOL! I really enjoyed this episode, I'd only every seen these in passing but it was cool to understand the process 😀 And they look very awesome.

  20. Not gonna lie, this guy was incredibly boring. Reminds me of my own classes of systematics and taxonomy, and this did nothing to make it any more interesting. He speaks constantly in jargon, sounds bored and annoyed at the questions, and did not provide any examples of exciting research. How disappointing.

  21. is it possible with birds, or would it be impossible with all the feathers? but then you do that with reptiles and it works despite the scales…?

  22. How long does each stage take generally? Because these are being absorbed by static (dead) things I feel that each stage would take a considerable amount of time. Plus what looked like a small ray of some description looked great, can we get a close up of that . It kinda reminded me of the old screen-savers of dancing lines.

  23. ver-tee-bral? car-ta-lag-enous? We learnt completely different pronunciations in med school. Isnt it vert-ter-bral like vertebra and car-ti-legenous like cartilage? or is it just me?

  24. Funny to see a technique I use in my lab shown on here. We study zebrafish fin regeneration and we use this staining to look at how fins regenerate.

  25. Humans (small parts of them at least) ARE cleared and stained in the process of creating histology slides.

  26. Isn't the word fish already plural lol I thought this was a educational channel or is it a joke idk I don't get it

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