394 GASOMETRIC-MANOMETRIC METHODS 



chloride. Saturate the soln. with a mixture of 5% carbon dioxide 

 and 95% nitrogen. 



PROCEDURE 



1. Pipette 1 ix\. buffer-substrate soln. into the bottom of a micro- 

 liter diver. 



2. By means of a pipette extending nearly to the surface of the 

 soln. in the diver, pass a rapid stream of 5% carbon dioxide in 

 nitrogen through the diver for 30 sec. 



3. Pipette 0.3 fxl. enzyme soln. into the bottom drop in the diver 

 and again pass the carbon dioxide-nitrogen mixture through the 

 dive]' . 



4. Place the paraffin oil seal in the neck of the diver followed by 

 the mouth seal. 



5. In a parallel fashion, prepare a control diver containing 0.3 

 jxl. inactivated enzyme or soln. without enzyme. 



6. Proceed with the diver technique as described on pages 342- 



382. 



note: According to the work of Linderstr0m-Lang and Holter (1942), 

 the relativel.y high permeabihty of paraffin oil and flotation medium to 

 carbon dioxide would make it advisable to use oil and medium which are 

 saturated with the carbon dioxide-nitrogen gas mixture. The apparatus 

 illustrated in Figure 132 would be useful for charging the divers. 



THIAMINE AND COCARBOXYLASE 



The method of Ochoa and Peters (1938) for the estimation of 

 thiamine and cocarboxylase depends on the stimulating effect the 

 compounds have on the decarboxylation of pyruvic acid by alkaline- 

 washed yeast. Employing the Warburg technique these authors were 

 able to determine down to 0.01 /xg. cocarboxjdase and 0.05 [xg. thia- 

 mine. By adapting this method to the Cartesian /A. diver, Westen- 

 brink (1940) increased the sensitivity to the measurement of 0.05 

 ni/xg. cocarboxylase and 0.5 m^ag. thiamine. The sensitivity of the 

 method "v^aries with the variety of the yeast and the manner in 

 which it is washed. The thiamine determination may be carried out 

 most suitably when the concentration of cocarboxylase present is 

 small compared to that of the thiamine. For instance, in one experi- 

 ment, thiamine had no influence on the carbon dioxide evolution 



