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



CULTURING GUINEA PIG SPLENIC TISSUE 



The procedures for preparing tissue homogenates, inoculating emhryonated eggs, and col- 

 lecting yolk sacs are described by McDade in this manual. Splenic tissue and yolk sacs are 

 inoculated to F-G and CYE agar according to methods described by Feeley et al. in this manual. 

 Isolates suspected of being LDB must be confirmed as such with tests described elsewhere in this 

 manual. 



Add 10 g soil 



to 



100 ml sterile distilled 



HjO containing 0.5% 



Tween 60. 



Agitate on shaker for 30 

 min.— let settle for 5-10 min. 



Discard 

 sediment. 



I 



Centrifuge I i 



supernatant also 

 400 X g for 5 min. i 1 



Discard 

 sediment. 



Use supernatant 



for 



primary analysis: 



screen by 



direct FA, 



plate to 



F-G and CYE agars 



Centrifuge supernatant 

 2900 X g for 30 min. 



Sediment 



+ 



10 ml sterile water 



Discard 

 supernatant. 



Determine CFU/ml 



Adjust to <106 CFU/ml 



Inoculate guinea pigs 

 3 ml/animal. 



Figure 2. Preparation of Soil Samples for Guinea Pig hioculation 



89 



