Cultural and Biochemical Characterization of the Legionnaires' Disease Bacterium 



safranin or carbol fuchsin for 1 min; rinse and air dry. Examine microscopically using oil 

 immersion. 



The LDB is a gram-negative bacillus having an appro-ximate width of 0.5-0.7 /jm and a 

 length that varies from 2 to 20 nm or more. The cells appear red when counterstained with 

 carbol fuchsin (Figs. 1 and 2) and light pink when counterstained with safranin. LDB cells 

 on initial culture and passage on MH-IH medium may appear slightly swollen in regions 

 having vacuoles that are stainable with the Sudan black B fat stain. 



Prepare the carbol fuchsin counterstain by mixing the two following solutions. 



2. Fat stain 



Flood heat-fixed smear with Sudan black B solution and allow to stand 5 to 15 min. 

 Drain slide and blot dry. Apply xylene to smear several times either drop by drop or by 

 immersing the slide into a staining jar containing xylene. Blot the smear dry and counter- 

 stain with safranin for 60 sec. Rinse with water and let air dry. Examine using oil immersion 

 (Fig. 3). 



Cells should appear pink and contain either blue-black or blue-gray droplets in areas 

 that were vacuolated in the Gram stain. 



Prepare the Sudan black B solution by dissolving 0.3 gm of Sudan black B in 100 ml of 

 70% ethyl alcohol and letting the solution stand overnight at room temperature. 



3. Gimenez stain 



The LDB can be demonstrated in an infected egg yolk sac with the Gimenez stain: the 

 LDB stains red, and the yolk sac tissue appears as blue-green background cells (Fig. 4). The 

 Gimenez reagents and procedure are described in the chapter by McDade. This stain can also 

 be used for impression smears and frozen sections (see chapter by Blackmon et al. in this 

 manual). 



4. Flagella stain 



Recently, tlagella have been observed (Berenice M. Thomason, personal com- 

 munication) on several strains of the LDB by using a modified Leifson tlagella stain (i) or a 

 simplified silver-plating stain for tlagella (y). 



C. Preliminary Identification 



Organisms that have the three characteristics described below and have a cut-glass appear- 

 ance on F-G agar should be strongly suspected of being LDB, and should be further examined by 

 procedures listed under Definitive Identification. 



1. Requirement for L-cysteine-HCl 



All strains of LDB examined to date require L-cysteine-HCI for growth. Therefore 

 organisms that grow on BA medium, which does not contain L-cysteine-HCl, are unlikely to 

 be LDB. Gram-negative bacilli that grow only on L-cyesteine-HCl-containing media such as 

 CYE, F-G, and MH-IH agars are possibly LDB. Examine these further for brown pigment 

 and yellow fluorescence production. 



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