Primary Isolation Using Guinea Pigs 

 and Embryonated Eggs 



Joseph E. McDade 



Tlie properties of the Legionnaires" disease bacterium (LDB) have already been described. 

 Briefly, the LDB is a gram-negative, non-acid-fast bacterium tliat does not grow on most bacterio- 

 logic media (7) but can be cultivated on enriched Mueller-Hinton (M-H) agar (9), F-G agar (J), a 

 semi-synthetic medium derived from the supplemented M-H formula, and charcoal yeast extract 

 (CYE) agar, recently developed for cultivating the bacterium {2). 



Tile LDB was originally isolated in guinea pigs from autopsy specimens and propagated in 

 embryonated hens' eggs (7). Later, after the organism was successfully cultivated /// vitro, com- 

 parison studies were done to evaluate the relative sensitivities of the various isolation procedures 

 (6). Although guinea pigs were found to be somewhat less sensitive than bacteriologic media for 

 isolating the LDB. the fact that these animals are relatively resistant to most other bacteria 

 enhances the likelihood of isolating the LDB from specimens that contain contaminants. Ad- 

 mittedly, the contamination problem with the //; vitro systems could be solved by the addition of 

 appropriate antibiotics (5. 12), but the fact remains that guinea pigs and embryonated eggs are 

 good systems for isolating the LDB, having been used successfully in the first isolations of the 

 LDB from clinical (7) and environmental samples (4. J J). 



The following sections outline the procedures involved in isolating the LDB, with particular 

 emphasis on using guinea pigs and embryonated eggs. Although the discussion is aimed specifi- 

 cally at isolating the LDB from human lung tissues, the procedures can be modified slightly and 

 used for other types of specimens. 



COLLECTION AND PROCESSING OF SPECIMENS (see Figure 1) 



Several tliumbnail-sized pieces of lung tissue should be obtained from the affected areas at 

 autopsy. At least one tissue sample should be placed in 10% neutral Formalin and used in 

 subsequent pathology studies and in direct fluorescent antibody (FA) tests (7) for the presence 

 of the LDB. Another piece of tissue should be stored at -70°C as reference material, and the 

 remaining tissue should then be taken or sent to the appropriate diagnostic laboratory for /// vitro 

 isolation attempts. 



If these initial isolation efforts are unsuccessful, //; vivo isolation can be attempted with 

 guinea pigs and embryonated eggs if appropriate facilities are available. If not, tissue suspensions 

 should be prepared and shipped to the appropriate reference laboratory in accordance with the 

 instructions in Part Four of this manual. Specimens received by the reference laboratory can be 

 safely held at -70°C until processed. 



A. Initial Processing (see Figure I ) 



(NOTE: All personnel who work with specimens suspected of containing the LDB should 

 wear surgical masks and gloves, and specimens should be processed in a biological safety cabinet.) 

 Grind a piece of lung tissue with a sterile mortar and pestle using alundum as an abrasive. 



70 



