270 A SYMPOSIUM ON RESPIRATORY ENZYMES 



enization because of its uniformity. With this latter method, over- 

 heating of the solution during the stirring should be rigorously 

 avoided. With minced tissue from the Latapie, two methods of 

 obtaining aliquots are commonly employed. The minced tissue may 

 be weighed out as such or stirred up in a cool solution and pipetted 

 out. My preference is for the latter method, which is a simple one if 

 a wide-mouth pipette is used. It is also much more rapid and yields 

 somewhat more uniform respiration in duplicates and triplicates. 

 With the other method it seems difficult to keep all the tissue under 

 the same conditions of cooling, a desideratum when things are hap- 

 pening as rapidly as they do in such a tissue. Furthermore, I have no 

 confidence in single experiments and so would urge that triplicate de- 

 terminations be carried out. 



Complicating Factors Arising between Removal of the Tissue and 

 the Experimental RMn.— During this inevitable unphysiological pe- 

 riod in which the tissue is anaerobic, many breakdown processes 

 occur. These lead to the accumulation of a number of metabolites 

 which can influence the results, especially during the early part of 

 the experiment. Whatever the speed of preparation, these changes 

 cannot be avoided. Those I have had to deal with have been the 

 accumulation of lactic acid, and striking changes in the hexosemono- 

 phosphates, adenosinetriphosphate, and phosphocreatine. The ac- 

 cumulation of lactic acid influences markedly the initial rate of res- 

 piration, and may suppress the effect of added lactate. In short 

 experiments it may lead to erroneous conclusions respecting the rates 

 of respiration of individual tissues. The literature contains not a few 

 instances of such misconceptions. It can be dealt with either by reduc- 

 ing the lactate content prior to the experiment, by aerating the tissue 

 in a Ringer solution long enough to bring the content to the normal 

 value, or by allowing the excess lactic acid to be dealt with in the 

 micro respiration vessels for an hour or so before the experimental 

 run. The former appears to be much less time-consuming and just as 

 effective. Oxygen is bubbled vigorously through the solution con- 

 taining the tissue. This is kept at room temperature. As regards 

 changes in the organic phosphate compounds during this interval of 

 preparation, it is very important to restore the normal relationships 

 existing prior to the experiment; otherwise the recovery process may 

 be taking place during part of the experimental period, confusing 

 the results of concomitant chemical balance experiments. This is 

 particularly important for muscle tissue, and it is necessary to de- 

 termine, for each tissue, how long an equilibration is required to 



