Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media 



The semisynthetic medium (Table 1) generally supported excellent growth of the standard 

 inocula described, although inocula with A's less than 0. 10 very often did not grow when initially 

 transferred to this medium. However, cells harvested from the primary passage to the semisyn- 

 thetic medium grew when transferred at lower cell densities. Growth on the chemically defined 

 medium occurred readily on the first transfer, but with subsequent passages growth occurred 

 sooner and reached higher densities on the chemically defined medium than on the semisynthetic 

 medium. 



In an experiment with the semisynthetic broth in tlasks, in shaking tubes containing various 

 volumes of liquid (5 to 15 ml), and under different conditions of aerobiosis induced by incuba- 

 tion with or without shaking, growth was obtained in tubes which were incubated with shaking 

 and had COj seals. No growth was observed in tubes incubated anaerobically. Tubes incubated 

 stationary with CO, seals did not contain significant growth until placed on the shaker, after 

 which their A's increased from 0.19 to 0.77. Tubes incubated stationary without COj seals had 

 no visible growth for several days but had final A's ranging from 0.48 to 0.61 after COj seals 

 were added and the tubes were again incubated with shaking. These results suggested that the 

 LDB was a strict aerobe, which required CO2 and ready access to oxygen, and that it was 

 potentially sensitive to excessive aeration. 



Adding agar to the chemically defined medium strongly inhibited growth of low concentra- 

 tions of inocula. Dilutions of inocula ranging from 10^' to 10"' grew in 7 to 10 days when 

 "plated" in broth in petri dishes and incubated in a 95% air-5% CO, atmosphere. Similar plates 

 containing Difco agar had no apparent growth at dilutions of more than 10^- . Others with 1% Ion 

 Agar No. 2 supported growth at 10"'', but when 1'? soluble or insoluble purified potato starch or 

 0.2% charcoal (Norit A) was added, the effect of the agar was completely reversed. The semisyn- 

 thetic agar and the F-G agar under moist, aerobic conditions supported comparable levels of 

 growth and cell yields, although the lag period on the semisynthetic medium was shorter. In air, 

 with a pH above 6.8, the growth rates (but not final cell yields) were definitely lower than those 

 obtained in candle jars. However, 0.1% alpha-ketoglutaric acid strongly increased the growth rate, 

 and cell yields were substantially higher at pH's above 6.8. Higher cysteine concentrations had no 

 apparent effect. The conditions required for growth raised many questions about the role of pH 

 and CO2 , the depletion of oxygen in a candle jar, the use of alpha-ketoglutaric acid as a source of 

 CO, , and the effect of pH on cysteine in the medium. 



These data did not clearly delineate the functions of the factors tested. More complete 

 experiments were conducted in air, air with COj added, and air with CO, removed, using media 

 containing low concentrations of casein hydrolysate. The effects of pyruvate and alpha-ketoglu- 

 tarate were also determined (Fig. 2). The results obtained in "air" were between the extremes 

 obtained with "added" and "removed" CO2 and are not shown in Fig. 2. At pH 7.2, no apparent 

 growth occurred in air or in air depleted of CO2 when neither pyruvate nor alpha-ketoglutaric 

 acid was added. The results also showed that at pH 7.2 without CO, , alpha-ketoglutaric acid and 

 pyruvate stimulated rates of growth and cell yields; however, the resulting cell yields were not 

 greater than 33'v-66% of that obtained when only CO2 was added. Carbon dioxide was clearly 

 required for growth at pH 7.2 and organic acids could not entirely replace it. Further, at this pH, 

 the growth rates and cell yields were significantly higher in the presence of pyruvate than in that 

 of alpha-ketoglutarate. 



However, at pH 6.5 with no organic acids added, CO, improved but was not required for 

 growth (Fig. 2). When the organic acids were present, cell yields in the absence of CO2 were only 

 66% of those obtained when CO2 was present. In the presence of 5%' CO; for an extended 

 growth period, the cell yields without added organic acids were only 85% of those obtained with 

 added pyruvate or alpha-ketoglutarate. These results indicate at least two separate roles for CO2 

 and the organic acids and emphasize that both are required for maximal growth. 



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