THE CRITICAL TIME LIMITS OF OPERATIONS 33 



fixed blocks as a standard for estimating roughly enzyme losses 

 caused by paraffin embedding, etc. 



The Effect of Embedding, etc. 



Since alcohol does not destroy alkaline phosphatase to a sig- 

 nificant degree, frozen sections from alcohol-fixed blocks may 

 be used to control the effect of later procedures. Sections were 

 cut from a paraffin block and incubated for 0, 10, 20, 40, 80, 160, 

 and 320 minutes in the glycerophosphate incubation mixture 

 given above. This series of sections was compared with a simi- 

 lar series prepared as frozen sections from an alcohol-fixed block 

 for the same periods. The comparison showed that a consider- 

 able loss of activity occurred during infiltration and embedding. 

 In a number of such experiments it was estimated that the loss 

 of enzyme activity may amount to 75 percent of the total. The 

 loss, however, did not appear to be localized but occurred to 

 the same extent, as far as may be distinguished by the eye, in all 

 sites in which the enzyme occurs. If the fixation, infiltration, 

 etc., were carried out under anaerobic conditions, a much greater 

 destruction of enzyme activity was found to occur. 



The Critical Time Limits of Operations 



Preliminary experiments suggested that loss of apparent 

 enzyme activity might be occurring in some stages of the pro- 

 cedure. Two groups of experiments were therefore carried 

 through to see to what extent this could be controlled. Three 

 blocks of material were fixed in 80 percent alcohol and then 

 taken to absolute alcohol after 2 hours. From one of these 

 blocks a series of frozen sections was prepared. The second 

 block was allowed to spend 2 hours in each of the dehydrating 

 and clearing agents and infiltrated with wax for 8 hours at 

 60° C. The third block was allowed to spend 24 hours in each 

 dehydrating and clearing agent and was then infiltrated for 48 

 hours at 60° C. When series of sections from all three blocks 

 were prepared by the glycerophosphate method, comparison of 

 the sections showed that a loss of about 50 percent of the enzyme 

 had occurred in both the second and third blocks (using the 

 frozen sections as standard) ; but there was no significant dif- 

 ference between the second and third blocks themselves. Thus 



