178 PRODUCTION OF CARBON DIOXIDE 



end two Pyrex glass tubes, in which are placed respectively the bacteria 

 to be used and the indicator, the change in color of which measures 

 the respired CO2' which is forced through the system. 



In order to facilitate experimentation, a non-pathogenic acid-fast 

 organism was used. This was Bacillus butyricus, obtained from the 

 stock culture of the Hygienic Laboratory, and isolated originally 

 from butter. The rapid growth of this organism is an advantage; 

 48 hour cultures grown on glycerin agar and incubated at 37°C. were 

 used for all the experiments. The heavy growth obtained was 

 washed off into a 0.75 per cent solution of dextrose in distilled water. 



As a basis of comparison, the same experiments were also per- 

 formed with Bacillus subtilis, a non-acid-fast organism, which had 

 been planted upon agar-agar and incubated for 18 hours at 37°C. 

 previous to use. These organisms were then transferred to a 0.75 

 per cent solution of dextrose in distilled water. 



For each experiment 2.5 cc. of 0.75 per cent dextrose solution con- 

 taining the organisms were used. The dextrose solution had a pH 

 of 7.0. The hydrogen ion concentration of the solution was changed 

 by adding drops of various concentrations of NaOH or H2SO4 from 

 a standard dropper. In solutions requiring a greater amount of 

 acid or alkali more concentrated solutions were used, thus keeping 

 the volume nearly constant. 



The NaOH was prepared from the best reagent obtainable, handled 

 with all necessary precautions to avoid the absorption of CO2, and 

 kept under soda-lime tubes. The H2SO4 was boiled for some time to 

 get rid of most of the volatile impurities. 



The indicators used for determining the pH were thymol blue, 

 brom phenol blue, methyl orange, methyl red, brom cresol purple, 

 phenolsulfonephthalein, and phenolphthalein. Buffer solutions, made 

 according to Sorensen's tables, were used as standards for 

 comparison. 



The rate is taken as the reciprocal of the time required for the 

 change in the pH value (in the indicator tube) from 7.8 to 7.6. This 

 time varied according to the amount of bacterial suspension used. 



The temperature was kept at 21° =h 1°C. 



Control experiments were performed with all the solutions in the 

 absence of bacteria, and also with dead bacteria killed by boiling 

 for one-half hour. 



