NO. 3596 ENZYME METHOD — TAYLOR 15 



with accompanying drop in pH and curtailment or termination of 

 digestive activity. 



The 30 percent solution has proven superior to all other materials 

 tested in speed of enzyme activity, in maintaining a relatively stable 

 pH, and in preventing or curtailing bacterial activity. 



Specimens containing glycerin, when cleared in this enzyme buffer 

 solution, produce a raj)id pH drop that may descend to the undesirably 

 acid pH 6.0 in less than one week if the glycerin is not carefully washed 

 from the specimen. 



Summary of the Clearing and Staining Method 



1. If specimen is not already in alcohol, preserve it in 10 percent 

 formalin for one week to 10 days. 



2. Soak all formaldehyde from specimen. 



3. Prepare a saturated solution of sodium borate in distilled water. 



4. Prepare a stock solution of one-half to one percent potassium 

 hydroxide in distilled water. 



5. Bleach specimen in about 10 percent stock hydrogen peroxide 

 and 90 percent stock potassium hydroxide solution. 



6. Prepare an enzyme-buffer solution of 30 parts saturated sodium 

 borate solution (supernatant) and 70 parts distilled water of volume 

 equal to 10 to 40 times that of specimen. 



7. Place specimen directly in buffer solution or, if necessary, remove 

 any large quantities of alcohol, water, or potassium hydroxide from 

 specimen by soaking it in a borax solution, and then place it in buffer 

 solution. 



8. Add about one-fourth teaspoon of enzyme to solution for small 

 specimens, slightly more for large specimens; mix and place aside to 

 clear at about 25° C. 



9. Change solution (steps 6 to 8) in one week to 10 days, and repeat 

 until specimen has only a few small areas of opaque muscle tissue 

 remaining. 



10. Stain specimen in a solution of stock potassium hydroxide and 

 alizarin dye. 



11. Remove viscera and undesired parts. 



12. Return specimen to digestion solution (steps 6 to 9) until 

 completely cleared. 



13 (optional). Remove oils by working specimen through alcohol 

 series into xylene. When oils have been removed, work back through 

 alcohol series and wash to remove all xylene. 



14 (optional). Dissolve dense deposits of guanin in a 2 to 4 percent 

 aqueous solution of potassium hydroxide. 



15. Work specimen gradually into full glycerin and add a few 

 crystals of thymol. 



