Primary Isolation Media and Methods 



James C. Fcclcy. George H'. Gurnnin. and Robert J. Gibson 



Rickettsial tecliniques were used by McDade et al. in tiie initial isolation of the Legion- 

 naires' disease bacterium (LDB) (4). In attempts to grow the LDB on artificial media, Dr. Robert 

 Weaver inoculated portions of suspensions of LDB-infected embryonated hens' egg yolk sacs 

 obtained from Drs. McDade and Shepard onto 17 different bacteriological agars. Of the media 

 tested, the only one that supported growth of the LDB was Mueller-Hinton agar, supplemented 

 with I'A hemoglobin and 1% IsoVitaleX (MH-IH) (/). 



After studying MH-IH agar, we developed two solid media that better support the growth of 

 the LDB from tissue. We also developed a diphasic medium for culturing blood. All these 

 media-Feeley-Gorman (F-G) agar (/), charcoal yeast extract (CYE) agar {2). and CYE diphasic 

 blood culture medium (.?)— contain chemicals necessary for growing the LDB. Soluble ferric 

 pyrophosphate or ferric nitrate replaces hemoglobin, and L-cysteine HCl replaces IsoVitaleX in 

 these new media. 



The CYE agar is the most sensitive artificial medium developed to date for isolating the 

 LDB, but it does not offer the differential clues for LDB colony identification that the F-G agar 

 allows. Consequently, we recommend that both CYE and F-G agars be prepared as described 

 below and used in combination. Although we no longer use MH-IH agar for isolating the LDB, we 

 include the instructions for preparing it as a reference. For blood cultures, we recommend a CYE 

 diphasic medium. 



Preparation of Media 



L MH-IH Agar 



Component A Mueller-Hinton agar 38 gm 



Distilled water 490 ml 



Component B Hemoglobin powder 10 gm 



Distilled water 490 ml 



Component C IsoVitaleX (#1 1876) 20 ml 



Prepare components A and B separately, and autoclave at 12rC for 15 min; cool to 50° C, 

 combine A and B, and hold at 50°C in a water bath. Prepare Component C by adding 10 ml of 

 sterile distilled water to each of two vials containing lyophilized IsoVitaleX. Add the contents of 

 both vials to the A-B mixture. Before pouring 20-mI quantities of medium into each of a series of 

 plastic dishes ( 1 5- X 100-mm), adjust the pH so that the final pH of the cooled medium will be 

 6.9. Allow agar plates to cool to room temperature. Measure the final pH of a plate of cool agar 

 (procedure in the Quality Control section of this chapter). The final pH must be between 6.85 

 and 6.95 in order to consistently support growth of the LDB. 



78 



