Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media 



On the other liaiui, the presence or absence of glucose in the semisyntlietic medium iuid no 

 effect upon growth. Although 1% glucose caused lactate to fomi in 0.8 to 0.^)-A/M/nil amounts, 

 no lactate was formed in the absence of glucose. No sugars other than glucose were tested. The 

 presence of amylose, amylopectin. dextrin, glycogen, yeast extract, or bovine albumin did not 

 affect cell yields. 



In the presence of 0.1% alpha-ketoglutaric acid, some acids, including 0.4% pyruvate, fuma- 

 rate, and succinate, had no effect on growth, but others such as citrate, acetate, and malate 

 totally inhibited growth; lactate slightly inhibited growth. The concentrations of alpha-ketoglu- 

 taric acid, pyruvate, and casein hydrolysate could be varied somewhat without affecting growth. 

 Nevertheless, during several experiments in which growth was marginal because of limited casein 

 hydrolysate or, in a medium which by itself did not support growth, 0.1% pyruvate stimulated 

 growth. In experiments with the chemically defined medium, very small but definite increases in 

 growth were observed when at least 0.1% of pymvate was added. 



The results with glucose, polysaccharides, and organic acids show that the bacterium does 

 not use these products as catabolic substrates for energy and carbon. The effects of alpha-keto- 

 glutaric acid and pyruvate are complex, although there is little question that they are beneficial 

 to growth. However, there is little evidence that they provide energy. It remains to be detennined 

 whether they are involved in buffering, transamination reactions, or are intermediaries or regula- 

 tors of some metabolic process. When the media were being prepared, we noted that both 

 alpha-ketoglutaric acid and pyruvate function as chelators because precipitates that fomied as the 

 pH was adjusted were reduced or eliminated. 



C. Nutritional requirements 



Cysteine was required for growth as previously reported (]0), and growth was directly 

 proportional to cysteine concentrations between 0.002% and 0.01% (Fig. 5); higher concentra- 

 tions did not increase cell yields and were slightly inhibitory. Cystine did not substitute for 

 cysteine, but glutathione did substitute for cysteine on an equimolar basis. Although the initial 

 growth was delayed with unadapted cells, cells adapted to utilizing glutathione had equal growth 

 rates and cell yields (Fig. 6). Under favorable growth conditions, the presence of 0.12 M dithio- 

 threitol, 2-mercaptoethanol, thioglycolate, or methionine moderately to completely inhibited 

 growth in medium containing only 0.02% cysteine. These and other results suggest that cysteine 

 functions as a required amino acid and is not required as a reducing agent or chelator. Indepen- 

 dent studies by one of us (JRG) showed that serine, methionine, arginine, valine, leucine, isoleu- 

 cine. and threonine are also required for growth of at least 10 LDB strains representing four 

 serogroups. 



When all the vitamins (Part I, steps 3-5, Table 1), starch, and oleic acid were removed, growth 

 was delayed. However, all further attempts with several strains to demonstrate a vitamin require- 

 ment have failed, although one of us (MWR) used these same media to demonstrate thiamine and 

 biotin requirements of Escliericlua coli. Similarly, others have been unable to demonstrate any 

 vitamin requirements in liquid synthetic media (W. J. Warren and R. D. Miller, personal commun- 

 ication). 



In addition to the -SH compounds above which inhibited growth, 1 /ig/nil of oleic acid, in 

 the absence of starch, completely inhibited growth; in the presence of 0.05% starch, 10 /jg/ml of 

 oleic acid did not inhibit growth. Whether the fatty acids of agars are inhibitors is not yet known; 

 nevertheless, it is known that 1% starch was optimal for agar media. The presence of 0.05% 

 ether-purified Tween 80 instead of starch totally inhibited growth, as did ethylenediaminetetra- 

 acetic acid. Inhibition by the latter compound plus the observations of inhibition by citrate, 

 acetate, and malate suggested that trace elements played a major role in metabolism. One of us 

 (MWR) demonstrated a requirement for iron in the defined medium with alpha-alpha dipyridyl; 

 other results indicate requirements for calcium and magnesium. 



36 



