MEASUREMENTS OF OXYGEN EVOLUTION 847 



sizing plant rapidly becomes surrounded by a layer of water that is alkaline, 

 deficient in carbonic acid and supersaturated with oxygen^three factors 

 each of which may strongly affect the rate of photosynthesis. 



Perhaps the most serious source of error is the fact, studied by I^iep 

 and Minder (1909), that the gas bubbles emerging from cut stems of water 

 plants contain not pure oxygen produced by photosynthesis but a mix- 

 ture of oxygen with air from intercellular spaces. The nitrogen content 

 of the bubbles varies with the rate of bubbling. As the air is drawn from 

 the intercellular spaces by the stream of photosynthetic oxygen, new air 

 diffuses into them from the medium, so that even after an extended period 

 of illumination the bubbles still contain some nitrogen. 



The precise manometric method was introduced into the investigation 

 of photosynthesis by Warburg in 1919 (see figure 25. 3 A). Since one 

 mole of carbon dioxide is consumed for each mole of oxygen liberated, the 

 manometric method can only be used if the carbon dioxide consumption is 

 completely or partially eliminated as a source of pressure changes. This 

 can be achieved by the use of a large volume of liquid in contact with a 

 small volume of gas, or by means of an alkaline buffer. In the first case, 

 the comparatively high solubility of carbon dioxide in water enables the 

 plants to use dissolved gas for a considerable time while only slowly re- 

 ducing the pressure of carbon dioxide in the gas phase. The use of buffers 

 enhances this stabilizing effect of the aqueous phase, though at the cost of 

 introducing a "nonphysiological" pH. 



In the application of manometric technique to respiration, practically 

 complete elimination of carbon dioxide as source of pressure changes could 

 be achieved by binding this gas chemically outside the cell suspension. 

 For this purpose, the reaction vessels were provided with a central well (or 

 side tube) containing alkali. A similar procedure cannot be applied to 

 photosynthesis because the latter requires the presence of carbon dioxide 

 in the suspension. The alternative of eliminating oxygen from the gas 

 phase (for example, by placing white phosphorus, or pyrogallol, in the 

 side tube) and measuring pressure changes due to carbon dioxide alone, 

 also does not recommend itself, because the rate of photosynthesis is sensi- 

 tive to oxygen pressure and often is strongly reduced by anaerobic condi- 

 tions (cf. chapter 13, page 326). Furthermore, elimination of oxygen 

 makes the application of the respiration correction difficult if not impossible 

 since in the dark, respiration is totally suppressed, whereas, in light, some 

 oxygen produced by photosynthesis is drawn into the respiration cycle 

 before reaching the external absorber. 



We are thus left with the choice of using cells suspended in a carbonate 

 buffer, thus working at a high "unphysiological" pH, or employing an acid 

 solution with an unphysiologically high carbon dioxide content (e. g., phos- 



