8 PROCEEDINGS OF THE NATIONAL MUSEUM vol. 122 



be cleared with materially less enzyme, but it is desirable to reduce 

 the volume of buffer solution if a very small amount of enzyme is 

 used. Mix well but do not froth, close the container, and set the 

 solution aside at 20°C to 30°C (most room temperatures are satis- 

 factory) to clear the specimen. Higher temperatures for digestive 

 activity are not recommended as they invite increased bacterial 

 activity and may denature connective tissue in lower vertebrates. 



Enzymes lose potency in the solution and should be added only 

 at the time they are to be used. 



6. Leave the specimen in digestion until one-tenth to one-fourth of 

 the muscle tissue remains. Change the digestion solution every week 

 to 10 days. If specimen has been in glycerin, the first digestion 

 solution must be changed m four or five days; the second and sub- 

 sequent solutions may be retained for a week to 10 days. 



The digestion solution may be cloudy (due to some enzyme 

 preparations) or clear, and it tends to remain so unless coagulated or 

 precipitated by some extraneous materials. It will darken gradually 

 with release of stains and pigment from the specimen. Clearing of 

 the specimen will first be noted along the edges of the body and in 

 the thinner portions such as the abdominal wall, the caudal peduncle, 

 and the limbs. The specimen will lose rigidity, becoming very 

 flexible as digestion proceeds. Some small specimens can be cleared 

 adequately in one or two days; larger specimens and those containing 

 enzyme inhibitors may require weeks or months in the solution to 

 become sufficiently cleared. 



Although a solution may remain active for several weeks, the 

 maximum activity appears to decline after a week to 10 days. As a 

 general rule, a solution should be discarded before it has been used two 

 weeks to avoid bacterial growth and excessive pH drop. 



The odor of the solution will initially be similar to that of trypsin 

 powder. Gradually, inoffensive odors due to the digestion may be- 

 come evident. Specimens that have been stored in such materials 

 as oil of cloves, isopropyl alcohol, and thymol, even though washed 

 to remove these materials, may impart their strong odors to the 

 solution. Growth of bacteria may result in very offensive odors of 

 putrefaction from the solution, in which case the solution should be 

 discarded. The specimen is then cleaned by placing it in 70 percent 

 ethyl alcohol or stock potassium hydroxide solution for a short time 

 and, beginning with step 4, is returned to a new digestion solution in 

 a clean container. If the odor of putrefaction is again produced, the 

 process must be repeated but an increase in the amount of satm'ated 

 borax solution is suggested (stock solution 3). 



When the specimen is nearly transparent — having lost three- 

 fourths or more of its dark muscle tissue and retaining only a few 



