NO. "596 ENZYME METHOD — TAYLOR 7 



and their organs loosened or spread to allow access of the bleachmg 

 solution. 



Bleaching at this time is important. If done later, the evacuated 

 body cavities tend to become filled with bubbles. Remove bubbles, 

 if possible, following bleaching. A small vacuum pump is useful for 

 this purpose. If the bubbles cannot be removed, they will slow the 

 clearing, but they will disappear in a closed actively digesting enzyme 

 solution in a few days. 



The enzymes do not extensively remove color pigment (chromato- 

 phores and guanin) from specimens, but dark pigment is noted to 

 flow out and materially darken digestion solutions subsequent to 

 partial bleaching. Complete dissolving of the pigment from a speci- 

 men, thus, may not be necessary. Bleaching is continued until all 

 dark areas have become decidedly pale, the time varying from about 

 half an hour for poorly pigmented specimens to several hours for 

 specmiens with thick layers of pigment (e.g., atherinid fishes). 



4. Place the specimen in a buft'er solution, adequate to cover it, of 

 volume 10 to 40 times that of the specimen. If the specimen has 

 been in glycerin, use the methods described in the section "Glycerin 

 Specimens" (p. 10). 



Large reservoirs of liquid, other than enzyme buffer solution, 

 which are in the evacuated l^ody cavities of a partially digested 

 specimen, should be diluted by placmg the specimen in a borax solution 

 for several hours prior to placing it in the enzyme buffer solution. 

 Excess water will dilute the bufl'er solution; hydroxides, isopropyl 

 alcohol, and ethyl alcohol wiU tend to elevate the pH of the solution 

 above the optimum. Traces of these liquids that may cling to the 

 outside of a specimen can be ignored. 



Excessive volume of the buffer solution wdll dilute and slow 

 enzyme acti\Tty. Insufficient volume will permit a rapid pH drop 

 and resist diffusion of digestion products from the specimen. The 

 adequate volume of buffer solution to use appears to be 10 to 40 times 

 the volume of the specimen. A minimum of 200 ml. of the solution 

 has been found satisfactory for very small specimens. 



Several specimens, if properly fixed, may be placed together in 

 one solution for clearmg. 



Glass and plastic containers both have been used for the process 

 ^nth good results. Containers that can be sealed are preferred as 

 they tend to limit bacterial contamination, permitting longer use of 

 a solution. 



5. Add the enzyme (trypsin poM-der). One-fourth of a teaspoon 

 (about 0.45 grams) of enzyme is adeciuate for most specimens weighing 

 up to 1 or 2 ounces and requiring 400 ml. of liquid or less for immersion. 

 Larger specimens may require more enzyme. Tiny specimens may 



