GROWTH REQUIREMENTS 



819 



atmosphere (0-03-0-04 per cent.). He found that if slopes of Br. abortus were 

 incubated in a glass jar containing 10 per cent. COg, good growth occurred in 24 

 hours, while under aerobic conditions there was no growth at all. Analysis of the 

 gas over a culture of B. subtilis revealed the presence of CO2, and it was therefore 

 concluded that the success of Nowak's method depended on the evolution of this 

 gas rather than on a decrease in the partial pressure of oxygen. Further work by 

 Smith (1924) and McAlpine and Slanetz (19286) confirmed 

 the importance of CO 2. Smith showed that development 

 was much better in an atmosphere of 10 per cent. CO, 

 than in sealed tubes, and that in agar shake cultures, either 

 sealed or incubated in an atmosphere of 10 per cent. CO2, 

 growth occurred, not in a band below the surface as Bang 

 (1897) and his co-worker Stribolt had found, but on the 

 surface itself. 



In spite of these observations, it was not easy to under- 

 stand why growth should occur in sealed tubes, or why, in 

 the absence of added CO 2, growth in shake tubes should 

 occur in a band below the surface. 



A fuller study of the gaseous requirements of Br. abortus 

 (Wilson 1931a) showed (1) that the organism would not grow 

 anaerobicaUy even in the presence of added COg, nor aerobically 

 in its absence ; (2) that growth would occur in partial pressures 

 of oxygen varying from 0-5-99-0 per cent., provided a minimum 

 of 0-5 per cent. CO2 was added, and in partial pressures of 

 CO2 varying from 0-5-98-0 per cent., provided a minimum of 

 0-5 per cent, oxygen was added ; (3) that the optimum partial 

 pressure of oxygen for development was about 21 per cent., 

 i.e., that normally present m the atmosphere, and of CO2 about 

 10 per cent. No evidence was obtained to suggest that a par- 

 tial pressure of oxygen lower than that normally present in the 

 atmosphere was beneficial to growth. It seemed clear, therefore, 

 that neither growth in sealed tubes nor the band phenomenon 



in shake tubes could be due to a preference of the organism for microaerophihc conditions. 

 Further observations (Wilson 1930) showed that CO2 was given off by biu-iung cotton- wool 

 plugs, and to a less extent by heated paraffin wax, rubber stoppers, and seahng wax. 

 Analysis of the gas mside sterile sealed tubes revealed the presence of COg in amounts 

 varying from about 1-3 per cent. — a proportion ample to initiate growth in inoculated 

 tubes. The larger the number of orgamsms inoculated, the less need was there for 

 additional CO2, since the organisms themselves produced a certain amount of this gas. 

 But with inocula of any size, growth was always most rapid and luxuriant when the 

 partial pressures of oxygen and COg most nearly approached the optima. 



Similarly, evidence was brought (Wilson 19316) to suggest that the band phenomenon 

 in shake tubes was due to the necessity of an adequate concentration of CO2 (Fig. 174). 

 It was found that this gas was given off to a certain extent by the orgamsms themselves, 

 and to a still greater extent by certain media, particularly those containing serum. Growth 

 could not occur at the sui'face, because the CO2 was given off into the atmosphere ; nor 

 could it occur in the depths of the medium, because the conditions were anaerobic. It 

 therefore commenced in a zone as near the surface as was consistent with the mamtenance 

 of an adequate partial pressure of CO2. If the tube was sealed, or was incubated in an 

 atmosphere of 10 per cent. CO2 then growth occurred at the surface, where the optimum 

 partial pressure of oxygen existed. This explanation, when shghtly amplified, was found 



Fig. 113.— Brucella 

 abortus. 

 (jllycerine agar slope 

 culture, 3 days, 37^ 

 C, in corked tube, 

 showing character of 

 growth on direct 

 isolation from the 

 tissues. 



