M. H. SOULE 263 



periment, 90 per cent CO, and 10 per cent O2 were used with good results. The high tensions 

 of CO2 necessarily produced marked changes in the acidity of the medium, but it was of 

 extreme interest to note the rich growths obtained under such conditions. In general, the 

 protozoa are unable to tolerate tensions of over 50 per cent of this gas. 



Of more interest, perhaps, than the action of high tensions of CO2 on the growth 

 of bacteria is the question of the absolute necessity of this gas for germ life. In all 

 probability a certain tension of this gas is required for the growth of some if not of 

 all organisms, and if this partial pressure is not available, the germs may die. Such a 

 concentration must be extremely small. From the fact that many of the reported ex- 

 periments designed to test this point cannot be successfully repeated by independent 

 investigators it seems probable that factors other than the presence or absence of free 

 CO2 have influenced the results. 



The removal of the free CO2 as fast as it is formed by a rapidly growing culture pre- 

 sents many difficulties. Any method used must of necessity be a continuous one to 

 prevent, if possible, the accumulation of the respired gas in close proximity to the 

 cells. In addition, the procedure must alter only the concentration of this gas, for if 

 changes are produced other than in the tension of CO2 the effects noted cannot con- 

 sistently be attributed to the presence or absence of this substance. 



The problem has been attacked in two ways: (i) the freshly inoculated media 

 (broth, agar slants, or agar plates) are placed in close proximity to an alkaline ab- 

 sorbent such as KOH to absorb the CO2 as fast as formed; (2) a rapid stream of CO2- 

 free air is passed over the cultures to remove mechanically the free CO2 respired by 

 the cell. In some of the reported experiments the two methods have been combined. 



The second method would seem to offer the most interesting possibilities, and it 

 has been extensively used, but the results, in so far as inhibition is concerned, have 

 been uniformly disappointing. If a stream of air is passed over an agar slant by lead- 

 ing the air inlet to the base of the agar, the passage of the air quickly desiccates the 

 medium. Novy and Soule found that, if the vapor pressure of the aerating gas was 

 adjusted to prevent the drying out of the medium, it was impossible to inhibit the 

 growth of the human tubercle bacillus, B. suhtilis, or other organisms. In fact, the 

 passage of 500 cc. per minute of COz-free air over the surface of freshly inoculated 

 agar slants or Kitasato plates gave better growth than in controls under ordinary con- 

 ditions. Analyses of loo-cc. samples of the gas after it had passed over the cultures 

 gave negative tests for CO2. However, these investigators found that by using 

 extract agar pH6.o in place of the usual beef infusion agar PH7.4 in their COi-free 

 aeration experiments, it was possible to inhibit the growth of, andeventually kill, 5. 

 typhosus, B. sultilis, and other organisms. 



The first method, in which alkaline absorbents are present to remove the respired 

 CO2, has received a great deal of consideration. Moore and Williams' placed soda 

 lime in the bell jars to remove the CO2 when testing the growth of the tubercle bacil- 

 lus in high oxygen tensions. Wherry and Ervin^ connected freshly inoculated tubes 



' Moore, B., and Williams, R.: loc. cit. 



^ Wherry, W. B., and Ervin, D. M.: J. Infect. Dis., 22, 194-97. 1918. 



