2 PROCEEDESTGS OF THE NATIONAL MUSEUM vol. 122 



The new method described in this paper substitutes enzyme diges- 

 tion for the alkaline maceration. It offers several advantages, includ- 

 ing ease of clearmg the old material. Other methods such as cleaning 

 by dermestid beetles or flesh maceration by boiling are usually not 

 desirable in the preparation of skeletons from small, delicate specimens 

 because the bones may be lost, damaged, or distorted. 



Purified trypsin powder was first tested, in distilled water with a 

 trace of potassium hydroxide, on formalin-fixed specimens that had 

 been stored for several years in ethyl alcohol. These tests were slow 

 in producing transparent specimens. The odors given off from the 

 solutions were very disagreeable but the results indicated that enzymes 

 should be explored. 



Because the initial preparations remained in good condition during 

 two years m glycerin, tests of various enzyme-buffer solutions were 

 undertaken. Of these, the sodium borate (borax) solution was found 

 to be superior. It supports the most rapid enzyme activity while 

 maintaining a relatively stable and desirable pH over a long period of 

 time, and it inhibits bacterial growth that results in disagreeable odors. 



Due to the great variation in specimens and in their preservation, 

 results are not always predictable. The enzyme method generally 

 produces good transparent study specimens from individuals that 

 have been properly preserved and cared for prior to enzjmie treatment, 

 except where thick or dense connective tissue is present as in some 

 small species and most large specimens. The enzymes are less harm- 

 ful to specimens than strong alkalis because the digestion removes 

 muscle tissue and stains and permits slight collapse of membranes 

 over the evacuated areas rather than extensive swelling with its 

 splitting and distortion or disintegration of tissue. Specimens will 

 fall apart occasionally or disintegrate during enzyme digestion as 

 well as in caustic potash solutions. This appears to result from 

 faulty preservation that permitted prior connective tissue alteration 

 or digestion. These specimens are more likely to be usable following 

 digestion than foUowdng caustic treatment because the former frees 

 the specimen of bulky and weighty materials that cause damage 

 when it is moved. 



I have used the enzyme method successfully to clear several hun- 

 dred specimens of fishes of more than 100 species, belonging to 40 

 families, and also several specimens of amphibians, reptiles, and 

 mammals. The method has been of value in restoring glycerin 

 specimens of fishes that had been treated previously with potassium 

 hydroxide. The enzyme method would thus appear to be useful as 

 a means of clearing small specimens of all vertebrates. 



I have not tested proteolytic enzymes other than those of pancreatic 

 origin. Their maximum activity is usually at a pH below 7.5 or in 



