Method for Isolating Legionnaires' Disease Bacterium from Soil and Water Samples 



Determine the total CFU/ml in tiie final suspension, adjust to 10*^ CFU/ml, and inject 3 ml into 

 each of two guinea pigs. Refrigerate the final suspension for 2 to 3 days until bacterial quantita- 

 tion is completed before inoculating the guinea pigs. 



If low 

 KIO^) 



Centrifuge 



100 ml sample 



2900 X g for 30 min. 



Discard 

 supernatant. 



Resuspend 



sediment in 



10 ml sterile HjO. 



Inject 

 3 ml/animal. 



If moderate 

 (104 to 106) 



Inject 

 3 ml/animal. 



If high 

 (>106) 



Dilute with 



sterile HjO 



to 106 CFU/ml. 



Inject 

 3 ml/animal. 



Figure 1 . Preparation of Water Samples for Guinea Pig hioculation 



Obsei-ving the Inoculated Guinea Pigs and Collecting Spleens. Measure the temperature of 

 the inoculated guinea pigs at a predetennined time each day for 7 days, and observe the animals 

 for signs of illness, i.e., ruffled fur, water eyes, prostration, and hypothermia. If there is a rise in 

 temperature of 0.6°C for 2 consecutive days or any rise in temperature with 1 of the above 

 symptoms, sacrifice and examine the affected animal. Sacrifice and examine all other guinea pigs 

 in the study at 7 days. Aseptically remove splenic tissue from the animals for culturing. Culture 

 peritoneal exudates and material swabbed from peritoneal walls by direct plating on CYE and 

 F-G agars. 



88 



