W. J. V. OSTERHOUT 173 



from neutrality may be compensated by adding CO2 to the tap water 

 or removing CO2 (by means of a current of air free from CO2 or by a 

 current of hydrogen). In this way the solution may be brought to 

 the most convenient pH value; all experiments are then started at 

 this value. It is desirable to choose a value in the region where the 

 indicator is sensitive. 



In case alkali is added to bring the solution to a given pH value it 

 must be done before the normal respiration is measured, so that the 

 buffer effect of the alkali will be the same in the measurements of 

 normal and abnormal respiration. 



The buffer effect of all reagents must be carefully measured. This 

 may be done by means of an apparatus recently described.^ In the 

 case of the reagents employed in these studies there is practically no 

 buffer effect (unless the contrary is expressly stated). The result of 

 buffer action is to make the amount of respiration appear smaller than 

 it really is. 



It was found that no alkali or acid (other than carbonic) was 

 given off by the organisms studied. This was determined by placing 

 the organisms in tap water free from CO2 and allowing them to respire 

 for some time so as to change the color of the added indicator; the COj 

 was then driven off (by a current of air free from CO2) and it was 

 found that the indicator returned to its original color. 



It was found by control experiments (in which the indicator was 

 added after respiration took place) that the indicator itself (at the 

 concentrations employed) had no effect upon respiration. 



The accuracy of the measurements depends on developing skill in 

 matching standard colors under uniform conditions. The standard 

 colors are prepared by making a series of buffer mixtures** containing 

 the same concentration of indicator as the experimental solutions and 

 contained in Pyrex tubes of the same size as those in which the or- 

 ganisms are respiring. Various devices may be employed to make 

 the matching more accurate. A background of white or light gray 

 paper is used by many, while others prefer opal or ground glass. In 

 some cases it is advisable to screen off the lower part of the tube or 



^ Osterhout, /. Biol. Chem., 1918, xxxv, 237. 



8 Cf. Osterhout, W. J. V., and Haas, A. R. C, Science, 1918, xlvii, 420, foot- 

 note 7. Frequent renewal of the bufifer solutions is advisable. 



