Physiology: Characteristics of the Legionnaires' Disease Bacterium 

 in Semisynthetic and Chemically Defined Liquid Media 



L. Pine. JR. George. M.W. Reeves, and W'.K. HarreU 



INTRODUCTION 



Recently, semisynthetic and cliemicaliy defined liquid media were described (6) for growing 

 the Legionnaires' disease bacterium (LDB). These media have allowed certain new obsei-vations 

 concerning growth characteristics of the organism and verification of characteristics observed 

 using the more complex Mueller-Hinton medium supplemented with hemoglobin and IsoVitaleX. 

 Feeley-Gonnan (F-G) agar, and charcoal yeast extract agar (CYE) (7. 2). In this chapter, we 

 discuss the media, the methods used for detemTining growth responses, certain idiosyncrasies 

 related to the use of the media, and some nutritional requirements and physiological aspects of 

 the LDB. 



METHODS 



A. Media 



The basic fomiula for the semisynthetic medium for growing the LDB is in Table 1 : that for 

 the chemically defined medium is in Table 2. A 2X concentrated broth medium was prepared by 

 combining double-strength solutions, adjusting the pH, and sterilizing by filtration (0.45 micron, 

 Millipore Corp.). The other solutions, which contained starch and oleic acid or agar, or both, 

 were prepared, autoclaved for sterilization, and added to the 2X broth medium while they were 

 hot to produce either the complete medium or a medium of 5/4 or greater concentration. Liquid 

 media were used within 24 h. Complete agar media were tubed at 10 or 15 ml; 8 and 1 2 ml were 

 used if they were at a 5/4 or greater concentration. These tubes or other basal agar media were 

 stored at 5°C for 1 to 2 wk. Before they were used, agar media were melted by being steamed for 

 5 min: if necessary, test materials were added to the melted agar. The melted media were then 

 poured into plates, which were always used within 16 h. Similarly, culture tubes (18 X 150 mm) 

 were prepared by adding 1 ml of water or chemical solutions to 4 ml of 5/4-strength basal 

 medium. 



B. Standard Inocula 



Most of our early studies (6) were done with LDB strain Philadelphia 1. which was isolated 

 from a patient who died in the 1976 epidemic of Legionnaires" disease (LD) in Philadelphia (3). 

 We later included other strains in all stages of media evaluation. Inocula for various experiments 

 were prepared as follows: cultures were grown on F-G plates or slants: cells were washed off the 

 agar surface with sterile distilled water and stored as thick suspensions (approximately 9.0 mg dry 

 wt of cells/ml) at -70°C. Media were inoculated with a freshly thawed cell suspension by placing 

 1-3 drops (0.05 to 0.15 mg dr\' wt cells) on agar plates containing 10 or 15 ml of medium or in 

 tubes (18 X 150 mm) containing 5 to 15 ml of broth. In broth, this inoculum was sufficient to 

 raise the zero time suspensions to an absorbance (A) of 0.05 (1 drop or 0.05 mg) to 0.20 (3 drops 

 or 0.15 mg) at 660 nm. Relationship of A to cellular dry weight was detemiined by suspending 



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