Method for Isolating 



Legionnaires' Disease Bacterium 



from Soil and Water Samples 



George K. Morris, Peter Skaliy. 

 Charlotte M. Patton and James C. Feelev 



INTRODUCTION 



Thirteen documented outbreaks of Legionnaires" disease (LD) iiave occurred in the United 

 States since 1965 (2). Epidemiologic investigations in several outbreaks indicated tiiat the etio- 

 logic agent was transmitted in air. Among the environmental sources implicated were water from 

 air-conditioning cooling towers or evaporative condensers (3) and soils disrupted by constmction 

 work and presumably transported as dust by air currents (7.6). In order to document environ- 

 mental sources of the Legionnaires" disease bacterium (LDB) microbiologically, we modified the 

 primaiy isolation system that utilizes guinea pigs and embryonated eggs (described by McDade in 

 this manual). We used these procedures to isolate the LDB from 38 of 176 environmental samples 

 of water and soil collected during 1 1 epidemic investigatons in 10 states (5, unpublished data). 

 All samples were subjected to two types of analyses: (a) primary evaluation using the direct 

 fluorescent antibody (FA) test and culture media and (b) guinea pig inoculation and evaluation. 



COLLECTION AND PRIMARY ANALYSIS OF SAMPLES 



Water or soil and other solid samples should be collected asceptically in chemically-inert 

 sterile containers and refrigerated until analyzed. Personnel analyzing these samples should wear 

 proper safety attire and manipulate the materials only in a biological safety cabinet. 



Primary analysis involves the direct FA test and primary culture on Feeley-Gomian (F-G) 

 and charcoal yeast extract (CYE) agars. The media are described by Feeley et al. in this manual. 



Direct FA. Screen samples with direct FA to detemiine which contain bacteria which stain 

 with the LDB conjugate(s) and are tluis most likely to yield isolates of the LDB when cultured. 

 Prepare smears of water samples and aqueous suspensions of soil and other solid material by 

 completely filling the circles on a microscope slide with the sample. Let the slides air dry, fix 

 with gentle heat and then with 10% neutral Formalin. The procedure is described fully by Cherry 

 and McKinney in this manual. 



Four serogroups of the LDB have been defined to date (4). Although ideally all specimens 

 should be screened for the presence of all known serogroups, appropriate FA conjugates must be 

 available in order to implement such a policy. 



Primary Culture of Water Samples. Prepare a water sample for direct culture on media by 

 making serial 10-fold dilutions to lO""* in sterile distilled water. Inoculate portions (0.1 ml) of the 

 undiluted sample and of each dilution to individual F-G and CYE agar plates, in triplicate, and 

 spread with a smooth glass rod. Incubate the plates at 35°C in air plus 2.5% COj . Examine plates 

 daily for growth. After 2 to 3 days of incubation, calculate colony fonning unit (CFU) value for 



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