NO- 3596 ENZYME METHOD — TAYLOR 9 



small areas of yellow or brown muscle tissue as remnants of larger or 

 thicker muscle masses — it should be stained (step 7). 



Specimens may not clear or may clear slowly because of enzyme 

 inhibitors. Common indicators of inhibition are a musty odor, due to 

 formaldehyde, or extensive bottom precipitate with the tendency of a 

 cloudy solution to become clear. A bath in stock potassium hydroxide 

 solution for one or two days is suggested to aid in removing the 

 inhibitor. This is followed by repeating the digestion process, be- 

 ginning mth step 4. The purpose of the potassium hydroxide treat- 

 ment is to remove water and alcohol-insoluble enzyme inhibitors. 

 Alternation of potassium hydroxide and digestion treatments may be 

 repeated until the specimen becomes adequately clear, processes that 

 may require several weeks or months to complete. 



Some specimens, typically large individuals but also some small 

 ones, have thick connective tissue and remain opaque even in liquids 

 with a high refractive index. They can be made usable by cutting 

 away the undesired connective tissue after completion of the clearing 

 and staining process. 



Poorly preserved specimens may quickly disintegrate. To stop or 

 slow the digestion, about 10 percent formalin may be added to the 

 solution, or the specimen may be transferred (carefully) to 70 percent 

 ethyl alcohol. 



Providing the solution has had little digestive activity and has a 

 relatively high pH, no damage to adequately fixed specimens results 

 from their retention in a digestion solution for several days or weeks 

 following clearing. Unless extensive digestion takes place, the 30 

 percent saturated borax solution is adequate to maintain a satisfactory 

 pH of 7.5 or higher for several weeks in the presence of the enzyme. 



7. Prepare a stain solution by mixing a very small quantity of 

 alizarin red S powder in sufficient stock potassium hydroxide solution 

 for specimen immersion. Add alizarin until a deep purple or the 

 desired color intensity is reached. Place specimen in stain solution 

 and let it remain until the bones have become adequately stained. 



No advantage is evident in using the specially prepared stain 

 solution described by Davis and Gore (1947, p. 8) or Hollister (1934, 

 p. 92). A solution once prepared may be retained and used to stain 

 additional specimens. 



An alcohol stain, prepared by dissolving alizarin red S in 70 

 percent alcohol and made slightly alkahne by the addition of a few 

 drops of stock potassium hydroxide solution will stain the specknens 

 that started to disintegrate in digestion without further extensive 

 harm. After staining, proceed Mith them to step 12. 



8. Remove specimen from stain solution, rinse in distilled water, 

 and then eviscerate, remove scales and all undesired parts. 



241-650—67—^—2 



