Enzymes 179 



Table 3, since many of the figures quoted are actually aver- 

 ages, computed from widely divergent data. Some of the dif- 

 ferences may be due to purely technical variations (tem- 

 perature; length of exposure to the fixative; size of pieces 

 used, and, consequently, rate of penetration by the fixative) 

 and also to the different types of tissue used. The latter point 

 is important, since the relative amounts of sensitive and re- 

 sistant enzymes may vary considerably in different tissues. 

 To mention only one example: v^hile a large percentage of 

 the alkaline phosphatase activity of liver tissue is due to un- 

 stable hexosediphosphatase, intestinal mucosa contains very 

 little, if any, of this enzyme. 



If the effect of various fixatives is tested in Coujard sUdes, 

 purified intestinal phosphatase being used as the enzyme, al- 

 cohol and acetone are found to cause relatively little loss of 

 activity (distinctly less than 50 per cent in 72 hours at 

 5° C); neutralized formalin destroys over 75 per cent of the 

 enzyme in less than 24 hours at room temperatue but has 

 very little effect at icebox temperature. 



Decalcification of tissues by the regular procedures v^ill 

 destroy all phosphatases. Lorch^^ and Creep, Fischer, and 

 Morse^^ report that small pieces of bone can be decalcified, 

 after alcohol fixation, in a citrate buffer of pH 4.4-5 (expo- 

 sure, several days). After decalcification, the tissue is reac- 

 tivated around pH 9 (barbital buffer), dehydrated, and em- 

 bedded. To v^hat extent the enzyme is preserved has not 

 been determined, but enough activity remains to permit 

 localization by the usual methods. 



Frozen sections show^ a higher activity than embedded 

 tissues (DanielU);^^ hov^ever, there is a danger of loss of en- 

 zyme by diffusion, since fixation in acetone or alcohol will 

 not render the enzyme completely and irreversibly insoluble. 



Embedding in paraflfin causes a further loss in activity, esti- 



28. Lorch, I. J.: Nature, 158:269, 1946. 



29. Creep, R. O., Fischer, C. J., and Morse, A.: Science, 105:666, 1947; 

 Creep, R. O., Fischer, C. J., and Morse, A.: J. Am. Dent. A., 36:427, 1948. 



30. DanieUi, J. F.: J. Exper. Biol., 22:110, 1946. 



