Physiology: Characteristics in Semisynthetic and Chemically Defined Liquid Media 



washed cells in 1*7 Formalin, measuring the A, diying aliquots for 48 h at 75°C, and recording 

 the dry weight (Fig. 1). All A's were measured in a model B Beckman spectrophotometer at 660 

 nm. 



C. Incubation 



For maintenance, cultures were incubated in candle jars or in 959^ air-5% CO2 . Experi- 

 mental agar cultures were incubated in air in moist chambers, in moist candle jars, in tiglitly 

 sealed chambers dried and depleted of COj by sandwiching the agar plates between open petri 

 dishes filled with NaOH pellets, in dessicators from which CO2 was depleted by the presence of 

 concentrated solutions of NaOH, or in tighdy sealed containers with an atmosphere containing 

 approximately 4.8?r CO, . The CO2 in sealed containers was generated by mixing 2 ml of 10% 

 Na2C03 /liter of internal volume with an excess of 2.5 N sulfuric acid. Growth on agar plates was 

 measured by adding 5 ml of distilled water or 1% Formalin to each plate and removing the cells 

 from the surface of the closed agar plate with a 1-cm wire brad cut from a paper clip. The wire 

 brad was placed on the agar and rotated by placing the plate on a magnetic stirring device. The 



Table 2 



Composition of a chemically defined medium (CDLp) for the 



Legionnaires' disease bacterium 



Amino acid-organic acid solution 



Add 5 g each of serine, alpha-ketoglutaric acid, and sodium pyruvate to 500 ml of water. Add 0.5 

 g of each of the 1 8 amino acids listed below and bring the flnal solution to 1 liter. 



L-alanine, L-aspartic acid, L-asparagine (monohydrate), L-arginine (monohydrochloride), L-histi- 

 dine HCl (monohydrate), L-isoleucine, L-glutamic acid, L-glutamine, glycine, L-leucine, L-lysine 

 (monoliydrochloride), L-methionine, L-phenylalanine, L-proline, L-threonine (allo-free), L-trypto- 

 phane, L-tyrosine, and L-valine. 



Basal medium 



1. Combme the following ingredients in the order shown: 

 250 ml of the 4X salts solution. Table 1 . 



200 mi of the amino acid-organic acid solution described above. 



10 ml of the vitamin solution. Table 1 . 



0.1 ml of the thioctic acid solution. Table 1. 



0.1 ml of the coenzyme A solution. Table 1. 



0.5 g L-cysteine HCl. 



0.5 g glutathione (reduced). 



1 ml of the minor element solution. Table 1 . 



2. Adjust pH to 6.3 with 20% KOH. 



3. Add 1 ml of the hemin solution. Table I. 



4. Adjust the pH to 6.5 with 20% KOH, bring the volume to 500 ml with distilled water, and sterilize 

 this 2X concentrated medium by filtering through a 0.45-;um membrane tllter. 



Final medium 



Bring the sterile 2X concentrated medium to final volume with sterile distilled water. For general 

 use, the final broth medium should contain 0.05%- soluble starch (Eastman, Mallinckrodt, or Baker), and 

 an agar medium should contain 1%. "Ion Agar" No. 2 (oxoid) and 1% soluble starch. 



30 



