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How To Enzyme Clear and Double Stain Fish Specimens
A - B: Dehydratate skinned and eviscerated specimen in 95-100% ethanol.
C: Stain cartilage in alcian blue solution.
D: Neutralize alcian stained specimen in saturated sodium borate solution.
E: Clear tissues in trypsin solution.
F: Tissues should be translucent enough to see backbone and ribs.
G: Stain bones in an alizarin solution.
H: All bone should be stained a deep red color.

Depending on age of specimens and previous preservation, best results are usually obtained with small individuals (10-15 cm SL), but clearing and staining of larger specimens is possible, especially if the fish has been recently preserved and is not too thick or fatty. Once the specimen has been fixed in formalin (10%), fully rinsed in water (2 days), and transferred to 70% ethanol the following protocol renders good results:

A) Remove both eyeballs, and remove the scales and skin from one side of the body. Open the abdominal cavity and remove the gut, gonads and liver.

B) Dehydrate fish in 95-100% ethanol overnight. For larger individuals you may want to change the ethanol once during the dehydration phase.

C) Place the specimen in a 2:1 solution of absolute ethanol:glacial acetic acid in which enough alcian blue powder has been added to give the solution the rich blue color indicated within the white square in the figure below. Leave the specimen in the alcian blue solution overnight to ensure a good take-up of stain by cartilage.

D) Remove specimen from alcian solution and place in a solution of saturated sodium borate overnight. Change the solution two or three time until the smell of acetic acid ("vinegar") is more-or-less gone (usually about three solution changes over a couple of days).

E) Place specimen in a 1:3 solution of saturated sodium borate solution:distilled/deionized water in which a small amount of powdered trypsin has been dissolved. The solution should have enough trypsin to give the appearance indicated within the white square in the image below. Usually about a quarter teaspoon of trypsin per 12oz of dilute borate solution. Addition of more trypsin is unnecessary and won't speed-up digestion of muscle tissue.

F) If possible keep the trypsin solution warm (about 75C) and change the solution every two or three days (more often if the solution turns greenish blue). To ensure that the acidity does not rise add a pellet of KOH at each change. Remove from trypsin once the specimen has become translucent enough that the backbone is visible when the specimen is held up to the light (as in figure F).

G) Place specimen into a 2% KOH solution in which a little Alizarin Red S powder has been dissolved. The solution should be a dark purple (as indicated in the white square in figure G ).

H) Leave the specimen in the alizarin solution overnight or until the bones have stained a deep red color. If the solution turns red (rather than purple) add a pellet of KOH.

I) Transfer specimen through a series of glycerin:2% KOH solutions starting with a 30% solution. Leave in 30% glycerin for 1-2 days, and change to a 50% glycerin solution. While in 50% glycerin exposure to direct sunlight and/or the addition of a few drops of 4% KOH solution can help bleach dark pigments that may remain. Leave specimen in 50% glycerin: 2% KOH solution for 3-4 days.

J) Transfer specimen to 70% glycerin (this time use distilled water rather than 2% KOH solution to make a 70% glycerin solution). Specimens can be stored in this solution. Addition of a crystal of thymol will help retard fungal growth during long-term storage.

Over time the tissues will continue to clear while in 70% glycerin so don't be too worried if the specimen is somewhat yellowish or opaque at the thickest part of the body - over time this will become increasingly transparent. Cleared and stained specimens can be dissected and their anatomy visualized while immersed in 70% glycerin.

NOTE: For the study of connective tissues, particularly ligaments and tendons cleared and stained specimens can be transferred to 70% ethanol and over a period of a few days the connective tissues will become white and clearly visible. The specimen can then be returned to glycerin and in a few days the connective tissues will no longer be visible.

© 2007 American Museum of Natural History Back to Ichthyology Department