MEASUREMENTS OF OXYGEN EVOLUTION 845 



termined by titration with potassium iodide and thiosulfate. If pyrogallol 

 or the leiico dyes (indigo white, leuco methylene bhie) are used for oxygen 

 determination, the progress of oxygen hberation can be followed colori- 

 metrically or spectrophotometrically. The same methods are applicable 

 to the com-ersion of hemoglobin into oxyhemoglobin — a method of oxygen 

 determination first introduced into photosynthetic studies by Hoppe- 

 Seyler (1879) and used more recently by Hill (1937, 1939). Osterhout 

 (1918) suggested the use of hemocyanine-containing crab blood (which be- 

 comes blue in the presence of oxj'gen) for the same purpose. 



Tw^o physiological methods of ox3^gen detection wei-e discovered by 

 Beijerinck (1901) and Engelmann (1881, 188G and 1894), respectively. 

 Beijerinck's method utilizes the bioluminescence of certain bacteria (e. g., 

 Micrococcus phosphorens) , wliich becomes visible in the presence of ex- 

 tremely small quantities of oxygen (5 X 10 ~^ mm. O2 pressure above the 

 liquid, or 1 X 10 ~* m./l. in water, according to Harvey and Morrison 

 1923). Engelmann's method utilizes motile bacteria (e. g., Proteus vul- 

 garis), which come to rest in oxygen-free medium, but begin to move about 

 in the presence of traces of oxygen. This method has been sharply criti- 

 cized by Pringsheim (1886) ; for an answer to this criticism, see Engelmann 

 (1887). With the help of motile bacteria, the photosynthetic activit}^ of a 

 single cell can be observed under the microscope. Luminescent bacteria 

 have been used, e. g., in attempts to decide whether single isolated chloro- 

 plasts liberate oxygen in light (c/. Vol. I, chapter 4, page 62). 



The chemical and biochemical methods are difficult to adapt to a con- 

 tinuous control of the rate of photos}Tithesis. Physicochemical methods 

 therefore early attracted the interest of workers in this field. In modern 

 quantitative studies of metabolic processes, manometric measurements 

 have acquired a predominant importance; biochemists have found that 

 almost every biochemical reaction can be conducted so as to cause absorp- 

 tion or liberation of a gas and this often ])rovides the best means of meas- 

 uring its rate. The reactions of hemoglobin with oxygen and carbon 

 monoxide were the first for which this method was developed bj^ Haldane 

 and Barcroft; applications to respiration and photosynthesis came 

 next. Since the time of Sachs (1864), a cmde method of measuring the 

 volume of liberated oxygen was known and widely used — "bubble count- 

 ing." In quiet sohitions of a given surface tension, the gas bubbles de- 

 taching themselves from the leaves have an approximately uniform size, 

 so that the rate of gas formation can be calculated by multiplication of the 

 number of bubbles formed per unit time by the volume of a single bubble. 

 This method is simple and sensitive, but obviously fraught with errors, 

 caused bj'' the differences in wettability of leaf surfaces, coalescence of small 



