Primary Isolation Using Guinea Pigs and Embryonated Eggs 



F. Staining Smears 



1. Allow tlie yolk sac smears to air dry. Heat fix. 



2. Stain the smears by the Gimenez method as follows; 



a. Filter working carbol fuchsin onto slide (tiirough Whatman No. 2 filter paper) and 

 let stand 1 to 2 m in. 



b. Wash slide thoroughly with tap water over sink. 



c. Cover smear with malachite green for 6 to '^ sec. 



d. Wash slide thoroughly with tap water. 



e. Cover smear a second time with malachite green for 6 to 9 sec. 



f. Wash thoroughly with tap water. 



g. Blot slide dry and examine stained yolk sac smears uilder an oil immersion objective 

 with light microscopy. 



3. In properly prepared slides, the LDB stains red. and the yolk sac tissue appears as 

 blue-green background cells. 



G. Confirming that Isolates are Legionnaires' Disease Bacterium 



1. After examining the Gimenez-stained yolk sac smears, select infected yolk sacs for 

 further study that contain 50-100 organisms/field { lOOOX). 



2. Thaw the test tube containing the selected yolk sac by placing it under running tap 

 water. 



3. Remove the yolk sac from the tube, aseptically grind it with alundum. and mix it with 

 sterile PBS to form a 5% suspension. 



4. Inoculate aliquots of the suspension onto TS agar, blood agar, F-G agar, and CYE agar 

 (see Feeley et al., this manual). Incubate F-G agar cultures at 35°-37° C under 2.5% CO2 . 

 Incubate the other types of cultures at 3 5° -3 7° C (not in a candle jar). Incubate all cultures 

 for 10 days. 



5. Test an aliquot of the yolk sac suspension by direct FA to determine whether the isolate 

 is an LDB. Draw the rest of the yolk sac suspension up into a Pasteur pipette, dispense it 

 into sterile, 1-dram vials, and store them at -70° C as reference material. 



6. The LDB should grow on F-G or CYE agar, but not on the other media listed above. 

 Growth observed on the other media represents isolates other than LDB. In order to give a 

 definitive report of the presence of the LDB, the isolate's growth and morphological prop- 

 erties must be confirmed as characteristic for the LDB, and its serologic reactivity in direct 

 FA tests with standard conjugates to the LDB must be appropriate. 



7. Bleed all surviving guinea pigs by cardiac puncture (with a I 2-nil syringe and an 18-gauge 

 IVi" needle") approximately 1 month postinoculation, and process the blood to obtain sera. 

 Test these sera, and the sera obtained prior to inoculation, for reactivity with standard LDB 

 antigens in indirect FA tests. Even if the LDB has not been successfully isolated with the 

 procedures already described, the seroconversion of guinea pigs to a standard LDB antigen 

 indicates that the LDB was present in the original patient sample that was submitted for 

 analysis. 



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