NO. 3596 ENZYME METHOD — TAYLOR 11 



completely cleared by enzyme digestion. These specimens are often 

 brown and opaque, especially after long storage in glycerin. Fre- 

 quently the skin has been split, the tissues are swollen, and parts may 

 remain attached only by narrow strands of tissue. With careful 

 handling and clearing, these poor specimens can often be made very 

 useful unless the fragmentation of connective tissue by the alkali 

 treatment has been extensive. In digestion, much of the bulky ma- 

 terial is removed and the swelling is replaced by the collapse of ele- 

 ments, which approxunates their normal position. I have cleared 

 many specimens successfully with enzymes, after they had been 

 macerated for varying lengths of time in potassium hydroxide solu- 

 tions ranging from 0.5 percent to 9 percent, without damage beyond 

 that due to the alkah. 

 The suggested procedure: 



1. For average specimens, dilute the original glycerin and thymol 

 mixture to one-half by addition of distilled water, het stand for 

 about half a day. Tough specimens may be removed from the 

 original glycerin directly to distilled water or borax; on the other 

 hand, very delicate and badly macerated specimens should have 

 their glycerin diluted more gradually. 



2. Place specimen in one or two (depending on size) changes of 

 distilled water or borax solution for one-half day or longer to remove 

 glycerin. 



3. Clear specimen by digestion, following the appropriate steps 4 

 through 12 as described in the preceding section, and repeat as nec- 

 essary. Do not retain the initial digesting solution longer than four 

 or five days as, owing to the presence of glycerin, the first solution 

 rapidly putrefies and becomes acid. Since nearly all of the glycerin 

 and much of the muscle tissue will have been removed in the first 

 digestion solution, the second digesting solution will be more stable 

 and may be retained longer. 



Reactions of the Pancreatic Enzymes 



The powdered enzyme preparation is usually slightly acid. The 

 proteolytic enzymes present are active from slight acidity to moderate 

 alkalinity, with pH 7.5 to pH 9.0 usually cited as the optimum for 

 trypsin and chymotrypsin. 



These enzymes break down proteins, especially denatured proteins, 

 converting them to polypeptides and amino acids; thus, denatured 

 connective tissue is digested. Elastin is not easily denatured but is 

 digested by elastase. For this reason, it is advisable to determine 

 that elastase has been eliminated from the pancreatic enzyme prep- 

 aration. The other important connective tissue component, collagen, 



