Physiology; Characteristics in Semisynthetic and Chemically Defined Liquid Media 



In tlie liquid semisyntlietic or chemically defined media, the standard inoculum will grow in 

 dilutions of ICT'' incubated 7 to 10 days in shallow layers in a CO, -enriched atmosphere. At a 

 dilution of 10"^ or 2 X 10"' (final A = 0.05), this inoculum will grow slowly in slanted tubes but 

 will not produce the maximal cell yields obtained in control shake cultures. However, inocula of 

 lower concentrations do not grow with shaking. Even when we steamed the broths for 5 min to 

 lower the redox potential (as might occur with large inocula), growth of low concentrations of 

 inocula was not stimulated by shaking. Without CO, seals, there was less growth, and heavy 

 brown pigments were formed which were associated with bacterium's stationary phase (Table 3). 

 This effect of air curbing growth was directly correlated with a pH rise from 6.5 to 7.9 during 

 incubation in air, whereas growth under the COj seal barely increased the pH from 6.35 to 6.45 

 (Table 3). Control studies showed that the CO, seals decreased the initial pH from 6.5 to 6.2 

 within 48 h. These results show that in part the CO, atmosphere functioned as a buffer to 

 maintain the initial pH in agar media. CO, may have also regulated metabolism. 



Using reagent-saturated filter paper discs on the surface of semisynthetic agar, we obsei-ved 

 no effects with succinate, fumarate, lactate, acetate, oxalate, gluconolactone, glucuronic acid, 

 glycerol, gelatin, and cobalamin. Yeast extract, pyruvate, and alpha-ketoglutaric acid stimulated 

 growth, whereas citrate strongly inhibited it. 



B. Detemiiiiation of substrate 



Having detennined conditions of growth and developed a basal semisynthetic medium which 

 satisfactorily supported growth, we examined the medium to detect components which acted as 

 substrate sources of energy. 



An examination of the semisynthetic medium for constituents which affect growth showed 

 that cell yields were proportional to the concentration of casein hydrolysate (Fig. 3). Of the 

 amino acids tested, the presence of histidine, threonine, tiyptophane, tyrosine, or serine gave 

 substantially increased cell yields, with serine causing the greatest increase (Fig. 4). Cell yield 

 were slightly higher in the presence of glutamine. lysine, or proline; cell yields were not appar- 

 ently affected by adding alanine, arginine, aspartic acid, cystine, glutamic acid, isoleucine, leu- 

 cine, methionine, and/or valine to the casein hydrolysate. Chromatographic experiments did not 

 show that any one amino acid disappeared during growth in 0.4% casein hydrolysate. These 

 results show that the above specified amino acids were major sources of energy for growth. 



Table 3 



Effect of air and air + carbon dioxide on growth and final 



pH in shake culture* 



s 



* Medium was the semisynthetic (SSLp) broth with 1.0% casein hydrolysate and an initial pH of 6.5. Cultures were incubated for 

 65 hat 37°C and shaken at 70 rpm. Carbon dioxide (CO, ) was generated with 0.3 ml of ]0';i Na^COj plus 0.3 ml 2.5 N HjSO, 

 per tube. 



34 



