diaphanisation?

[PA]

I am not sure how I missed this post three years ago, but the actual method is usually referred to as clearing and staining a specimen. Potassium hydroxide or enzymes are used to clear the specimen after skinning and then various stains used to color the bone and sometimes the cartilage, the muscles are again cleared, and then the entire specimen is moved into glycerine and can be storred indefinitley. Here is a short bibliography of various papers used in clearing and staining.

There are some links listed on this post with some cleared and stained specimens.
http://www.taxidermy.net/forum/index.php/topic,199024.0.html

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Chapter VII.  CLEARING AND STAINING OF SPECIMENS
 
The majority of papers in this section pertain to clearing and staining of amphibians, reptiles, and fish, where this method is commonly used to observe skeletons, cartilage, and other systems.  In birds, clearing and staining is used primarily for embryo or nestling study.  

Bock, W. J., and C. R. Shear.  1972.  A staining method for gross dissection of vertebrate muscles.  Anatomischer Anzeiger, 130:222-227.

Burdi, A. R.  1965.  Toluidine Blue - Alizarin Red S staining of cartilage and bone in whole-mount skeletons in vitro.  Stain Technology, 40:45-48.

Campbell, S. C.  1986a.  A method for clearing and staining small fishes, amphibians and reptiles.  Pp. 29-32, in Proceedings of the 1985 Workshop on Care and Maintenance of Natural History Collections (J. Waddington and D. M. Rudkin, eds.), Royal Ontario Museum Life Sciences Miscellaneous Publications.  121 pp.

Campbell, S. C.  1986b.  A bibliography of clearing and staining small vertebrates.  Pp. 115-116, in Proceedings of the 1985 Workshop on Care and Maintenance of Natural History Collections (J. Waddington and D. M. Rudkin, eds.), Royal Ontario Museum Life Sciences Miscellaneous Publications.  121 pp.

Cumley, R. W., J. F. Crow, and A. B. Griffen.  1939.  Clearing specimens for the demonstration of bone.  Stain Technology, 14:7-11.

Davis, D. D., and U. R. Gore.  1947.  Clearing and staining skeletons of small vertebrates.  Fieldiana Technical Bulletin no. 4.  16 pp.

Dingkerkus, G., and L. D. Uhler.  1977.  Enzyme cleaning of Alcian Blue stained whole small vertebrates for demonstration of cartilage.  Stain Technology, 52:229-232.

Evans, H. E.  1948.  Clearing and staining small vertebrates, in toto, for demonstrating ossification.  Turtox News, 26(2):42-47.

Filipski, G. T., and M. V. H. Wilson.  1984.  Sudan Black B as a nerve stain for whole cleared fishes.  Copeia, 1984:204-208.

Filipski, G. T., and M. V. H. Wilson.  1985.  Staining nerves in whole cleared amphibians and reptiles using Sudan Black B.  Copeia, 1985(2):500-502.

Freihofer, W. C.  1966.  The Sihler technique of staining nerves for systematic study especially of fishes.  Copeia, 1966(3):470-475.

Freihofer, W. C., L. J. V. Compagno, and W. Rogers.  1977.  Additional notes on the use of the Sihler technique of staining nerves of small, whole specimens of fishes and other vertebrates.  Copeia, 1977:587-588.
 
Green, M. C.  1952.  A rapid method for clearing and staining specimens for the demonstration of bone.  Ohio Journal of Science, 52(1):31-33.

Hardaway, T. E., and K. L. Williams.  1975.  A procedure for double staining cartilage and bones.  British Journal of Herpetology, 5(4):473-474.

Humason, G. L.  1979.  Animal tissue techniques.  Fourth edition.  W. H. Freeman & Co.  San Francisco.  569 pp.

Lansdown, A. B. G.  1968.  A silver impregnation-toluidine technique for the demonstration of embryonic skeletal structures in paraffin sections.  Histochemie, 13:192-195.

Mayorga, H.  1965.  A rapid method for clearing and staining amphibian skeletons.  Journal of the Ohio Herpetological Society, 5(1):23-25.

Russell, E. L.  1973.  Improved methods for staining bones of small fetuses and vertebrates in Alizarin Red S.  BioScience, 23:366-367.

Russell, F. E., and R. G. McCandless.  1954.  Cleaning and staining the articulated skeleton in fishes.  Turtox News, 32(12):222-224.

Simons, E. V., and J. R. Van Horn.  1971.  A new procedure for the whole-mount Alcian Blue staining of chicken embryos, adapted to the clearing procedure in potassium hydroxide.  Acta Morphologica Neerlando-Scandinavica, 8:281-292.

Taylor, W. R.  1967a.  An enzyme method of clearing and staining small vertebrates.  Proceedings of the United States National Museum, 122(3596):1-17.

Taylor, W. R.  1967b. Outline of a method of clearing tissues with pancreatic enzymes and staining bones of small vertebrates.  Turtox News, 45:308-309.

Taylor, W. R., and G. C. Van Dyke.  1986.  Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage.  Cybium 1985, 9(2):107-119.

Wassersug, R. J.  1976.  A procedure for differential staining of cartilage and bone in whole formalin-fixed vertebrates.  Stain Technology, 51(2):131-134.

Watson, A. G.  1977.  In toto.  Alcian Blue staining of the cartilaginous skeleton in mammalian embryos.  Anatomical Record, 187:743.

Williams, T. W.  1941.  Alizarin Red S and Toluidine Blue for differentiating adult or embryonic bone and cartilage.  Stain Technology, 16(11):22-25.

Zug, G. R., and R. I. Crombie.  1970.  Modifications of the Taylor enzyme method of clearing and staining for amphibians and reptiles.  Herpetological Review, 2(3):49-50.

[Baccus]

Hi,

I am actually preparing to do this in the coming semester.  I need to wait for the hood at school to free up and then order the chemicals. 

I do not have the list in front of me, but the dyes are variable.  Classically the bones are red and cartilage is turned blue, but there are a few options there.  Send me and email, and I will forward the pdf files I have.  Or I will post the witches brew when I find it. 

If you are close to Rutgers NB, I will be using the bio lab there for this project.  Feel free to tag along.

Mark
notechy@gmail.com

[cyclone]

Hi,

I am actually preparing to do this in the coming semester.  I need to wait for the hood at school to free up and then order the chemicals.  

I do not have the list in front of me, but the dyes are variable.  Classically the bones are red and cartilage is turned blue, but there are a few options there.  Send me and email, and I will forward the pdf files I have.  Or I will post the witches brew when I find it.  

If you are close to Rutgers NB, I will be using the bio lab there for this project.  Feel free to tag along.

Mark
notechy@gmail.com

Isn't science grand!

FYI,  from past experiences I'd suggest ordering the chemicals before the fume hood becomes available..Be ready to rock as soon as it is clear....


and..You could take us all along if you'd just be kind enough to post a few pics of your progress..

[Alpinist]

There is a good introduction to the technique in chapter 4.2 of:
Hildebrand, Milton. 1968. Anatomical Preparations. Univ. of California Press. 100pp.

It's available in Google Books here: http://books.google.com/books?id=GhLs1n6t3JkC&pg=PA43&dq=anatomical+preparations+hildebrand&source=gbs_toc_r&cad=4

[Baccus]

Science is grand.  I am a Biomedical Engineer, but I dabble in bone and chemical work on the side. 

I was given a pair of baby cotton top marmosets in partial payment for a 15' anaconda mount (the guy still owes me the rest of the money, and anything else that dies in the zoo but worst case scenario, I will have a 15' snake in the collection for free).  There are also a few pygmy chameleons and other lizards waiting for this. 

I will post pictures as things come together. 

Thanks,
Mark

[grygon]

Would love to see this!

Am I correct that I see carolina has some staining kits to buy?  How dangerous are these chemicals? 

[PA]

The danger is not that great - at least the way I did the process many years ago. Museums do this on a regular basis - at least until CT scans became the norm. If you use the Potassium hydroxide method, the chemical is about as dangerous as Drano which I believe is Sodium hydroxide. You can neutralize the chemical prior to disposal - or use it to clean your sink drain on the way out. The dyes are only needed in very small quantities, and glycerine is essentially inert by itself. I did not bother with a fume hood as there are no noxious fumes in clearing and staining. You would want a hood for fixation in formalin.

We have quite a selection of many specimens done over the years, probably since the 1950's and they are truly neat preparations. You can use any fluid preserved specimen for the procedure but fresh specimens tend to fall apart, so I would suggest you first preserve the specimen in formalin in the position you wish it to be in, the use a series of washes to move into alcohol and then begin the procedure.

[Baccus]

Sorry for the delay, but I apparently lost the pdf files when my laptop crashed recently.  I just got another copy from my adviser. 

I am more than happy to forward them to anyone who is interested. 

[Damon]

Is it even possible to clear and stain animals much bigger that small lizards/mice/fish? I thought it only really worked well on small specimens due to the amount of fat in the tissue, bone density and so on.

[Alpinist]

You're right Damon. It only works well on small specimens.

To see some truly outstanding examples of the technique look up the artist Iori Tomita:
http://trendland.net/when-fish-meet-artistic-science/
http://www.designboom.com/weblog/cat/10/view/15095/iori-tomita-new-world-transparent-specimens.html

[Guus]

That's funny, I came across Tomita's website when I realized I had read something about that technique on here. So I searched for the thread only to find that Alpinist already posted some of his work. Anyway, here is his website, amazing stuff: http://www.shinsekai-th.com/en/top.php
I have a mole like this myself and when people see my collection they always want to know more about that skeleton.