NO. 350G ENZYME METHOD TAYLOR 3 



strong acids, which may be damaging to skeletal tissue. The pan- 

 creatic extracts are inexpensive, breaking down denatured proteins, 

 especially those of muscles, and work best in a favorable, slightly 

 alkaline medium. 



There are few published accounts on the use of enzymes in the 

 preparation of osteological material. Moser (1906: summarized in 

 Piechocki, 1961, p. 285) recommended a solution of potassium car- 

 bonate and trypsin powder for clearing embryos. The method has 

 remained relatively unknown. In my tests, clearing was slow, solu- 

 tions rapidly became acid, and there was loss of bone minerals. 

 Piechocki (1961, p. 228) summarized reported uses of papain, pepsin, 

 and trypsin for cleaning flesh from skeletons. The papain and pepsin 

 methods involve the use of acids and were not tested because of 

 probable damage to bones. The trypsin methods include the use of 

 sodium sulphide and higher temperatvires than I have employed. 

 My results indicate that sodium sulphide-enzyme solutions have 

 less advantage than borax-enzyme solutions for both clearing and 

 defleshing preserved specimens. Some damage to specimens occurred 

 from rapid pH drop to acid conditions. True (1948) described a 

 slightly acidified pepsin solution for removal of fats and proteins 

 before staining. 



I made frequent checks on the pH of all solutions during the 

 process of working out the method. The readings were obtained 

 from a Beckman pH meter. Model G, calibrated at pH 7.0 by use of 

 the "Fisher Standard Buffer Solution" specified as pH 7.00 ±0.02 at 

 25°C (monopotassium phosphate-sodium hydroxide bufl'er, 0.05 

 Molar). In comparative tests, specimens of one species with pre- 

 sumed identical preservation were used, and specimens of equal sizes 

 were selected insofar as possible. They were placed in solutions of 

 equal volume with similar enzyme content. All reactions were at 

 room temperature, which varied from 22°C to 27°C. 



I am especially appreciative for the laboratory assistance of Ken- 

 neth Karb, Phyllis Herrington, and Fanny Phillips, and the en- 

 com-agement received from the staff of the Fish Division in the 

 development of the process. 



Materials Required 



1. Alizarin red S powder. This material should be one of those 

 certified by the Biological Stain Commission as satisfactory for 

 staining bone. A few grams will be sufficient for several years use in 

 most work. 



2. Potassium hydroxide (KOH), U.S. P. grade. The pellet form is 

 the most satisfactory in rapid preparation of solutions. 



