Cleared and Stained Diaphonized Fish | Mikolji

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Cleared and Stained Diaphonized Fish

By Ivan Mikolji / 2014

I consider myself a person who is always trying to think “beyond the aquarium.” I travel far to swim with rare fish, meet ichthyologists, collect rare voucher specimens and field data for science, but most of all I try to fuse my interest in the aquarium hobby with science and art. I have always also been a very curious and creative person, and as a youngster I channeled all my creativity towards creating small living worlds inside an aquarium. My curiosity lead me to read books about fish and art but I never thought or did not know how to blend all my interests together. I remember exactly when it suddenly dawned on me that there was a whole world out there that nobody talked about. Typical aquarium books and pet shops were only the tip of the iceberg of the aquarium hobby. Anyways, that is a long story for another day. What I do want to show you is how incredible one of those “beyond the aquarium” places can be.

Cleared and Stained Diaphonized Hatchetfish Gasteropelecidae DSC05012

Once upon a time, many, many years ago I was in the Fish Collection ichthyologic lab of the Central University in Caracas, Venezuela. I was new to all the “science” behind tropical fish. I felt like I was in Geniusville, what NASA would look like if it worked with fish instead of rockets. The smell of formaldehyde, ethanol, and alcohol used to preserve specimens filled the air making a peculiar impression that still lasts till today. Every time I smell that particular scent it sends me back to a fish lab. The curator had let me in the lab and told me to wait until he finished something he was working on and could not wait. I stood in the middle of the room, my head resembled an oscillating fan, scanning everything from side to side, looking at all the bones, jars filled with thousands of preserved fish, microscopes, and dissecting equipment. I probably had my eyes and mouth wide open, too.


Cleared and Stained Diaphonized Silver Dollar Fish Metynnis mola DSC05756

On my right, I saw many shelves filled with hundreds of small plastic drawers. With my super curious nature, I could not resist being in the room without knowing what was in them. As I opened one of the small drawers, my eyes opened even wider and my jaw dropped. My expression at the moment could be easily compared to the one we all have seen on TV, when the pirate opens the treasure box filled with gold and diamonds. This was my first encounter with cleared and stained fish. The fish looked like red and blue gummy candy! How could this be? How did they do this? Every small drawer I opened had a different fish species. There were hundreds of drawers! Each drawer had a little tag on the outside with a number, scientific name, number of specimens in the drawer, approximate dimensions of the specimens, and the place where the fish were collected. Most of the scientific names on the tags were new to me. I scanned the tags until I recognized one, Serrasalmus irritans. I opened the small drawer and there were many specimens of this beautiful piranha.

The Diaphonization process is applied to smaller forms of animals and is commonly referred to as "Clearing and Staining." The specimens in the collection ranged from 2cm to 15cm long. The people in the lab referred to them in Spanish as “transparencias” = transparencies, but told me that the correct term was diaphonization. The process helps scientists see the skeleton structure of animals as it renders the flesh invisible, the bone (Calcium) red, and the cartilage blue. Clearing and staining a fish helps scientists compare the bone structure between different fish species. This is extremely helpful when two animals are thought to be different species but look very similar in the outside.  Sometimes a fish species can only be told apart from another similar species by counting the amount of spines or rays on a determined fin or by morphological differences in some of their bones. Clearly, if specimens have a constant and obvious difference in bone structure, it can be said that they are a different species. The process is not a standard when describing a new species but if it is implemented it gives extra support to the description or investigators work. The diaphonized samples differ from x-rays in that they can be manipulated. The specimens can be separated using tweezers and each bone can be examined under a microscope. Some of the drawers in the collection contained only little bits of bones, if you have patience for puzzles you could put the fish back together again. I asked, Prof. Francisco Provenzano, “Do you know where each bone goes or belongs on this Neblinichthys roraima, Punk Pleco?”, “Of course!” he answered (Thought in my mind… “That is so cool!”).


Cleared and Stained Diaphonized Red-bellied Piranha, Pygocentrus nattereri DSC05454



One of the things that came to my attention right away was that piranhas seemed to have a row of teeth hiding under the regular functional teeth. When I asked if this was a specific trait of piranhas, I was told that it was a trait shared by characins, it’s just more obvious in a piranha than in a small tetra because their teeth are bigger. When a tooth is lost, the one underneath is ready to take its place. They are replacement teeth. I really did not know that piranhas could replace their teeth until I saw the diaphonized specimens.


Cleared and Stained Diaphonized Angelfish Pterophyllum altum DSC05318

The diaphonization process is quite unique. It uses many chemicals which can be bought in a pharmacy or chemical store. It is important to handle all these chemicals correctly and with caution. The process is a question of trial and error. It takes practice and experience in order to get the results you want. The clearing and staining process can be resumed into 10 steps. I will breeze you through the process and give you a general idea of how it is done.

1.      Preservation: The specimens should be preserved using formalin for at least 5 days. Then, placed in an alcohol solution of different concentrations for up to 10 days.

2.      Gutting: The specimen’s guts should be removed.

3.      Dehydration: The specimens are placed in an ethanol solution from 2 to 7 days depending on the size of the fish.

4.      Staining the cartilage: The specimen’s cartilage are stained by placing them in alcian blue stain for 1 to 2 days.

5.      Neutralization: Specimens are placed in saturated sodium borate.

6.      Bleaching: Specimens are depigmented in hydrogen peroxide and potassium hydroxide solution for an hour or less.

7.      Clearing 1: Specimens are placed in a sodium borate and trypsin solution for about 7 to 20 days.

8.      Bone staining: Specimens are placed in an alizarin red dye and potassium hydroxide solution for about 1 to 3 days.

9.      Clearing 2: Specimens are placed back into trypsin solution for a week or so until the flesh seems almost invisible.

10.   Final storage in Glycerin: Specimens are placed in glycerin with thymol as a preservative for final storage.


Another thing that drew my attention was that some specimens were totally clear with no color, others only red, others totally blue, and others red and blue. After driving the people in the lab insane with so many questions, I got very good answers to why the difference in colors. First, if an investigator is only interested in seeing the skeleton, which he stains red, the blue part of the process is omitted simply because it is not needed. In other situations, the investigator has many samples of the same species so he can choose to dedicate a sample per render. He then stains one whole fish skeleton red, another whole fish can be rendered only for cartilage, which renders blue. A third sample could be rendered using both techniques and get a blue and red fish. The fish that are dark in color are probably small juvenile specimens whose calcium concentration is not optimal for the process and the outcome is not as great as in more mature specimens. The outcome also depends on how the fish was manipulated since the moment it was caught. If the fish is damaged in any way while it’s being caught, you will wind up seeing the broken bones, just as you do in an x-ray. If a fish is fixed using incorrect concentrations of formaldehyde or left too long in the preservation solutions you will have a different outcome when it is diaphonized. You also have to be very cautious and not leave the fish too long in any of the clear and staining solutions because that will affect the outcome, as well.

If you think about it, the diaphonization process sounds much like an art. It could be compared with the art of processing film photography, where you get different outcomes depending on how long the photograph is exposed to the different chemicals. It could also be compared with the art of cooking were fish would be the main course and every stage counts and affects the outcome. In my point of view, they represent a magnificent work of art from both nature and the scientists who applied the process. They resemble from colored x-rays to colored candy, intriguing the mind with magnificent detail of the morphological complexity. I am in awe at how these creatures were created.

Diaphonized specimens are also an important part of natural museums in their sense of reservoirs of knowledge. Cleared and stained specimen collections are tangible bone structure archives. The specimens can be accessed by investigators giving them a sense of what, where, and when something existed. They are always there, available for verification and study by the investigating community and why not, the curious community, such as myself.


This article has been published at:
• Practical Fishkeeping Magazine - January 2015


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