Biological Nitrogen Fixation 113 



tion (15), or to discuss the amicable outcome of that dis- 

 agieement. The important points to emphasize here are: 

 (a) the type of experiment that led to the discovery of ex- 

 cretion was implicit in the extremely primitive apparatus 

 a plant physiologist ordinarily worked with even as late 

 as twenty-five years ago; and (b) the exploitation of the 

 discovery by Virtanen and his collaborators through de- 

 velopment of a more ingenious technique was a noteworthy 

 triumph not to be overlooked. In comparison with modern 

 methods, the tool they forged may look disarmingly frail 

 for the task assigned, but it was a considerable advance 

 over anything else available at the time. 



Case History II. Iniiial Studies on Enzymafic 

 Pathways 



Studies at the University of Wisconsin at this time had 

 taken an entirely different direction both in technique 

 and goal. Impressed with the success of the microrespi- 

 rometer recently introduced by Warburg and Barcroft for 

 the study of enzyme mechanisms, and following the lead 

 of Meyerhof and Burk in applying this instrument to 

 biological nitrogen fixation, we attempted to study the 

 properties of the enzyme system responsible for nitrogen 

 fixation in red clover. Our version of a ''Warburg" ap- 

 paratus for study of plants is shown in Figure 3. Red clover 

 plants were grown in nine-liter Pyrex bottles on sand pro- 

 vided with the usual plant nutrients except combined 

 nitrogen. The bottles were fitted with appropriate acces- 

 sories so that the gas atmosphere furnished the plants was 

 controlled. Some plants were inoculated with the proper 

 bacteria and thus depended on nitrogen fixation for their 

 development; control plants were furnished ammonium-N 

 or nitrate-N. During the summer months when the plants 

 grew best, temperature was regulated by keeping the 

 bottles in a wooden "bath" through which water flowed. 



