NO. 3596 ENZYME METHOD TAYLOR 13 



described by Gomori, and many chemicals used in specimen preser- 

 vation, were made by me. None showed the digestive activity and 

 pH stability of a 30 percent saturated borax solution, many developed 

 strongly offensive odors, and some appeared to be moderate to strong 

 enzyme inhibitors. Very little digestive activity was observed in 

 solutions with pH readings above 10.0, but with a gradual drop in 

 pH the activity often increased. 



Although sodium chloride is an elastase inhibitor (Mandl, 1961, 

 p. 233), it slowed the reaction rate, and no effect on connective tissue 

 from its use was observed. Distinct loss of alizarin staining minerals 

 from fin rays was observed after digestion in the following (all with 

 rather rapid drop to low pH readings, the initial pH followed by final 

 pH in parentheses) : dilute trisodium phosphate (10.3 to 4.8) ; dilute 

 hexamine (hexamethylenamine) (7.4 to 5.5); dilute sodium hydroxide 

 (various dropping to 5.0-6.0); distilled water (6.4 to 5.2-6.0); 0.3 

 percent potassium carbonate, recommended by Moser (1906) for 

 clearing embryos by trypsin digestion (10.2-10.5 to 5.6-6.5); dilute 

 sodium sulphide recommended by Piechocki (1961, p. 228) for 

 defleshing specimens (10.0 to 6.5). Similar specmiens in solutions 

 with pH readings remaining above 7.0 did not undergo this loss. 

 No significant damage to connective tissue by digestion was observed 

 in specimens previously soaked in potassium or sodium hydroxide, 

 but many specimens that had been preserved and allowed to remain 

 in unbuffered formalin exhibited loss of the alizarin staining minerals. 



Specimens removed from formalin preservative without washing 

 digested very little, if any; and, in borax solutions, the enzyme 

 coagulated and precipitated at the bottom. These specimens could 

 be cleared adequately only after repeated changes of digestion solution 

 or after being washed in water for several days to remove the formal- 

 dehyde. Specimens that had remained in formalin storage for a 

 lengthy period of time generally took longer to clear than did speci- 

 mens stored in alcohol although the formaldehyde had apparently 

 been removed by soaking in water. LikeA\dse, specimens in alcohol 

 solutions that contained a slight odor of formaldehyde required pro- 

 longed clearing. 



The presence of 0.033 percent formalin in the borax buffer solution 

 prolonged the clearing tune for a specimen several times that of 

 solutions without formaldehyde, requiring several changes of digestion 

 solution, but 0.1 percent or more formalin in the buffer (in which 

 formaldehyde odor could not be detected) appeared to prevent all 

 digestion. 



Sodium borate solutions were prepared by using measured quantities 

 of borate and distilled water or by saturating distilled water with 

 borate powder and diluting wdth distilled water at room temperatures 



