Cultural and Biochemical Characterization of the Legionnaires' Disease Bacterium 



2. Brown pigment 



Inoculate either an F-G agar plate or slant, and incubate at 35°C in air plus 2.5% CO2 . 

 Pigment should be observable in areas of heavy confluent growth after 2-3 days of incub- 

 ation. Browning may also be produced with other media enriched with L-tyrosine at a 

 concentration equivalent to that found in Mueller-Hinton agar (1). Browning is not observa- 

 ble with CYE agar. 



3. Yellow fluorescence 



Inoculate a tube of F-G broth heavily with the suspected organism. Incubate at 35°C in 

 air plus 2.5%' COj . Examine daily in the dark with a 366-nm ultraviolet light. Broth should 

 tluoresce bright yellow within 3-4 days of incubation and continue to do so with additional 

 incubation. F-G broth has the same formulation as F-G agar except that it lacks agar-agar, 

 and the concentration of soluble ferric pyrophosphate (wt/vol) is 100 mg/liter. Tube broth 

 in 2-ml aliquots in screw-capped tubes (13 x 100 ml). 



D. Definitive Identification 



The LDB characteristically gives negative reactions for most biochemical tests with the 

 exceptions of catalase. oxidase, and j3-lactamase. Therefore, an isolate can only be definitively 

 identified as LDB with results from the direct fluorescent antibody (FA) test, gas liquid chroma- 

 tography (GLC), and deoxyribonucleic acid (DNA) hybridization. 



1. Direct FA test 



Prepare smears of the candidate organism, and stain it with all available conjugates 

 according to the methods described by Cheriy and McKinney in this manual. 



2. Gas liquid chromatography 



Prepare extracts of the candidate organism by the methods described by Moss et al. in 

 this manual, and examine for cellular fatty acids. 



3. Deoxyribonucleic acid (DNA) hybridization 



Brenner et al. have used this test veiy effectively for studying the LDB. Their proce- 

 dure is described in this manual. However, because this test is very specialized and time-con- 

 suming, it should be reserved for isolates that ( 1 ) have a compatible cellular fatty acid 

 profile by GLC and do not stain with direct FA conjugates prepared against all established 

 serogroups or (2) are considered to be LDB but have some unusual characteristic. 



BIOCHEMICAL CHARACTERIZATION 



This section is for those investigators who wish to further characterize an isolate as the LDB. 



A. Carbohydrate Utilization 



The LDB will not grow in the commonly used carbohydrate test media. For this reason, 

 carbohydrates were incorporated into especially prepared basal media to demonstrate utilization 

 by the LDB. All 15 strains tested grew but did not produce acid (Riley, Weaver, and Feeley, 

 unpublished data). Similar results were obtained with "rapid sugar" techinques (2). One test that 

 can be easily perfomied is Starch Utilization. Heavily inoculate an area of an F-G agar plate and 

 incubate for 3-5 days at 35°C in air plus 2.5%' CO2 . Flood plate with Gram's iodine and observe. 

 Most of the agar should stain blue because of the reaction of the starch contained in F-G agar 

 with the added iodine. There should be a clear zone void of blue color extending about 4 mm 



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