Cultural and Biochemical Characterization 

 of the Legionnaires' Disease Bacterium 



Robert E. Weaver and James C. Feeley 



Although few cultural and biochemical characteristics have been defined, they are suffi- 

 ciently distinct to enable a clinical microbiologist to differentiate the Legionnaires' disease bac- 

 terium (LDB) from other bacteria. Over 50 strains of LDB have been isolated and identified with 

 the tests described here. 



CULTURAL CHARACTERIZATION 



Inoculate the suspected isolate of LDB on charcoal yeast extract (CYE), Feeley-Gorman 

 (F-G), and blood agar (BA) media (4). In addition, the medium on which the LDB first grew, 

 Mueller-Hinton agar supplemented with \9r hemoglobin and 1% IsoVitaleX (MH-IH), may be 

 inoculated (optional). (The chapter by Feeley et al. in this manual describes these media and 

 their fomiulation.) Streak the inoculum on each medium to obtain well-isolated and discrete 

 colonies. 



The LDB grows best under aerobic conditions or in air plus 2.57c COj , depending on the 

 medium used. It does not grow anaerobically. Incubate all media except CYE at 35°C either in 

 air plus 2.5% COj or in a candle extinction jar. Incubate CYE agar at 35°C aerobically in a moist 

 chamber. Examine all plates daily for 2 wk. 



A. Colonial Morphology 



On the MH-IH agar, the LDB grows slowly. The colonies of the various strains which have 

 not been transferred more than a few times on the agar are pinpoint in size at 3 days. By 5-7 

 days, well-isolated colonies may reach a diameter of 3 or 4 mm. The colonies are convex, circular, 

 and have an entire edge. They are gray and glistening. In contluent areas the growth appears to be 

 slightly moist. 



On CYE agar growth of the LDB is apparent in 48 h or less in confluent areas at 35°C and 

 42°C. More growth occurs at 35°C than at 42°C. At 25°C. growth is apparent in 4-7 days. 

 Further description of growth on F-G and CYE agars is in the chapter by Feeley et al. 



B. Microscopic Morphology 



Remove a portion of the growth from the CYE agar with a bacteriologic needle. Emulsify 

 the growth in approximately 1.0 ml sterile distilled water to make a slightly turbid cell sus- 

 pension. Transfer a small aliquot of this suspension to each of two microscope slides, spread into 

 a film, let air dry, and then heat fix. Stain the smears using the Gram and fat stain procedures 

 below. 



I. Gram stain 



Flood smears with crystal violet solution for 1 min; rinse with water. Add Gram's 

 iodine for 1 min; rinse and decolorize with 95% ethyl alcohol. Counterstain either with 



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