194 STUDIES ON RESPIRATION. Ill 



ing the same concentration of phenolsulfonephthalein as the experi- 

 mental solution and contained in Pyrex tubes of the same size. The 

 tap water was found to have a pH value of 8.3. 



All experiments were done in Pyrex tubes each containing a total of 

 5 cc. of liquid as follows: Various amounts of tap water plus three 

 standard drops of bacterial emulsion and sufficient saturated solution 

 of ether in tap water to make the following concentrations of ether by 

 volume: 0.037, 0.183, 0.329, 0.438, 0.95, 1.46, 2.9, 4.38, 5.84, and 7.3 

 per cent. 



Each tube, therefore, contained a total volume of liquid amounting 

 to 5 cc. in which the concentration of indicator was the same. The 

 number of bacteria was made as nearly equal as possible in the dif- 

 ferent tubes by taking a uniform emulsion, mixing it thoroughly be- 

 fore taking the drops from it, and using a standard dropper so as to 

 have the drops of equal size. The emulsion was transferred almost 

 simultaneously to all the tubes. The tubes were then quickly closed 

 (by clean corks boiled in paraffin) with the exclusion of all air and 

 determinations were made by comparison with the standard set of 

 buffer solutions contained in tubes of the same size. In every case 

 there was a control consisting of organisms in tap water without 

 ether, and of ether in tap water without organisms. 



To see if phenolsulfonephthalein is toxic to bacteria, several trials 

 were made by placing bacteria in a liquid of known pH value and 

 after respiration had gone on for a definite time adding the indicator, 

 and comparing that result with the color of a control tube to which 

 the indicator was added at the beginning of the experiment. No dif- 

 ference in rate of respiration was observed.^ 



It was also found that ether has no buffer action to interfere with 

 the measurement of the rate of respiration. 



That the bacteria under these conditions give off no alkali or acid 

 (other than carbonic) is shown by driving off the CO2 at the end of 

 the experiment. The solution then returns to the original pH value. 



A great deal of variation in cultures of different ages was ob- 

 served. Those more than 24 hours old have a markedly lower rate 



^ For other details see Paper I of this series (Osterhout, W. J. V., /. Gen. 

 Physiol., 1918, i, 171. 



