Clearing and Staining Small Vertebrates
By Ernie Brown
Here’s a pdf copy to download: Clearing and Staining Small Vertebrates
When teaching the skeletal system in anatomy class or comparing the structure of homologous organs, it is useful to be able to make comparisons between skeletal features of different vertebrate organisms. The bones to be studied can be carefully dissected away from the muscle tissue and then reassembled, requiring a great deal of teacher or student preparation time. By use of the clearing method, however, the entire skeleton can be studied in relation to the muscles, circulatory system, and other organ systems. After a little experience with the procedure, it is a simple matter to introduce modifications which will improve the quality of the preparations. At times, two specimens, treated in a similar manner, will respond differently.
Fresh specimens may be preserved in 10% formalin solution or 75% alcohol for a period of three to five days. Generally, specimens fixed in formalin are more easily controlled and require less watching. Formalin fixed tissues often require a longer time for the clearing process and the specimen may not clear as completely as alcohol fixed specimens. A little experimentation will provide the best results for the type of specimens you wish to clear.
Completely eviscerate the specimen if the skeletal system is to be demonstrated.
Place the specimen in 2% KOH (potassium hydroxide) or 2% NaOH (sodium hydroxide). This step will decolorize the tissue and make it jelly-like in consistency. Use the same clearing solution for both alcohol and formalin fixed specimens. You can control the rate of tissue maceration by adjusting the temperature of the KOH or the room temperature. As the pigments are dissolved out of the tissue, the solution may become discolored, in which case it should be changed as often as necessary.
Staining the skeleton begins after the flesh has become cleared and the bones are visible through the surrounding tissue. Alizarine red S is the dye recommended by most authors but others also have used indigo-carmine, Bordeaux red, alizarol black3G, and alizarine black SBB. Make a saturated stock solution of alizarine red S and add it to the 2% KOH solution to make it reddish in color. Cover the specimen with the dye solution and leave it to set.
The specimen will usually stain completely within two days but additional time may be required for larger specimens. After staining, the specimen is placed in clear 2% KOH solution and destained until the muscle tissue is again clear and the stain remains only in the bones. If the bones were not stained completely, repeat the staining process and clear as before.
When the specimen looks nearly transparent, place it in glycerine for storage. This will complete the clearing process. The change from 2% KOH to pure glycerine must be gradual for large specimens but is not important with smaller ones. A half and half mixture of pure glycerine and 1% KOH is recommended for those large specimens.
When clearing fish, the scales must be removed in order to see the skeleton. Scaling is best done after the fish has been stained since the scales will be red and easily visible. Some lizards must also be scaled. The scales may be easily removed by gentle scraping in a dish of water with a wire loop or other similar instrument. The hair of mammals and the feathers of birds may best be removed prior to staining.
• Davis, D. Dwight and V. R. Gore. Clearing and Staining Skeletons of Small Vertebrates. Chicago Nat. Hist. Mus. Technique Ser., 16p. 1947
• Evans, Howard E., Turtox News, Vol. 26, No.2, February 1948
• Mayorga, Horacio. A Rapid Method for Clearing and Staining Amphibian Skeletons Journal of The Ohio Herpetological Society, Volume 5, Number 1, May 1965