Diaphonization absolutely fascinates me. Juxtaposing clear flesh and brightly-dyed bones, suspending a small thing in glycerine until it floats and almost appears to take on a new life… It’s something I’d love to try, if I were to stumble on a relatively fresh, small specimen that’d died of natural causes.
Also known as “clearing and staining” a specimen, diaphonization is a process in which a (generally small) specimen has its soft tissue rendered transparent while the bony structures are chemically dyed. It’s a way of preserving a specimen and producing a visually striking wet mount that illustrates the fully articulated skeleton without the inherent headache (and room for error) of cleaning, degreasing, and articulating a dry skeleton.
How is this achieved? Step by step, the process goes something like this protocol adapted from Dingerkus and Uhler’s “Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage”:
- Preserve fresh specimens in formalin to prevent decay. This involves allowing the specimen to soak in a 10% solution for at least two to three days.
- Rinse the specimen by soaking in several changes of fresh water (distilled or RO) for another day or two.
- Allow the specimen to soak in a solution made up of alcian blue, ethanol, and acetic acid for approximately eleven hours. This will stain the cartilaginous structures.
- Rinse specimen by soaking in two successive changes of 95% ethanol, for one to two hours each. Follow by soaking it in several dilutions of alcohol, from 75% down to 15%, for one to two hours each (or until the specimen sinks in the solution). Follow this with two to three hours in distilled water.
- Soak specimen in a solution of sodium borate, distilled water, and trypsin. Solution should be changed every two to three days, or sooner if it begins to turn a bluish color from the Alcian blue stain. Continue soaking until the bones and cartilage are plainly visible.
- At this point, some specimens are treated to a twenty four hour bath in potassium hydroxide and Alizarin red. This will stain bones a distinct purple or reddish color. Some specimens are treated only with Alizarin red, skipping the Alcian blue stain.
- Transfer through successive dilutions of potassium hydroxide and the preservative the specimen will be stored in (most likely glycerine with thymol), before placing it in its final container filled with the storage medium.
While beautiful, diaphonization is notoriously finicky– even small specimens can take a long time to clear and stain. In the interim, decay may set in. Alcian blue only works in an acidic environment, which may cause larger specimens to begin to break down long before the dye is ever able to reach the bones or cartilage. Specimens also need to be kept in preservative if they are going to be kept from decaying after staining. While this isn’t much of an issue with, for example, a frog or a small snake, finding a jar large enough for an animal over a foot long could prove problematic.
Diaphonization is far from a purely decorative art– while the specimens it produces are visually striking, they also serve the important purpose of offering a look at the internal structure of animals that may be too small to dissect. This means that things like skeletal deformities and birth defects can be seen in situ.
Interested in examining some diaphonized specimens? Fortunately, you don’t have to go to a natural history museum to do it. Several skilled sellers (like Lithographica on Etsy) have continued this combination of art and science, producing beautiful, display-worthy, ethically sourced specimens.