Indirect ImimiimnLiorcscencc Test tor Lefioniiaircs" Disease 



suspended in 0.01 M phosphate buffered siiUne, pM 7.6 (PBS). Make subsequent doubling 

 dilutions through 1:2048 in plain PBS. (We use Linbro Seientifie Co. rigid polystyrene 

 U-bottomed microtitration trays and a Cooke Engineering Co. automatic diluter. We place 

 0.01 ml of serum in a test tube containing 0.15 ml of NYS suspension and make subsequent 

 doubling dilutions in wells of the microtitration tray using plain PBS and 0.05-ml volumes. 

 Alternatively, replace the automatic diluter with hand diluters, or make all dilutions in test 

 tubes.) 



2. Using a Pasteur pipette, place a drop of each serum dilution from 1:64 to 1:2048 

 (or 1:256 if sera are being screened first) in its well of fixed antigen on a slide. Incubate 

 the slide in a moist chamber (e.g., petri dish containing moist filter paper) at 37°C for 

 30 min. 



3. Rinse slide quickly with PBS and place in a PBS bath for 10 min. Remove from the 

 bath and gently blot dry. 



4. Place a drop (approximately 20 ^1) of FITC4abeled antihuman immunoglobulin on 

 each well. Incubate as above for 30 min. 



5. Rinse slide quickly with PBS, place in a PBS bath for 10 min, rinse quickly with water, 

 and gently blot dry. 



6. Add several drops of buffered glycerol (1 part 0.5 M carbonate-bicarbonate buffer, 

 pH 9, in 9 parts glycerol) to the slide and mount with a 24- X 60-mm coverslip (No. I). 

 (Check the pH of the buffered glycerol mounting fluid frequently; the tluid should not 

 be used if below pH 8.) 



7. Read slides on a tluorescence microscope (see below), 



8. The serum titration end point is the highest dilution of serum that gives \+ fluorescence 

 (see below) of at least 507r of the bacteria estimated per microscopic field. The titer is the 

 end-point dilution factor. 



C. Controls 



1 . The primary control is a human serum having a specified titer against tiie serogroup 

 antigen used. For the test to be considered valid, the titer obtained with the control serum 

 should not vary more than 2-fold from the titer specified for that serum. 



2. The second control is PBS substituted for serum, to test for nonspecific binding of the 

 conjugate. If bacterial cells are visible, fluorescence intensity must be <\+ (see below). 



Legend for color plate: 



Indirect IF test of patient's serum for antibodies against Legionnaires' disease bacterium, serogroup 1 

 (Philadelphia 1 strain). Dilutions of serum and resulting fluorescence intensities are given under each 

 photograph. Staining endpoint = 1 + . Titer = endpoint dilution factor = 512. Magnification = 315X. 

 Note that autofluorescence (yellow-brown) of organisms at dilution of 1:1024 must not be interpreted 

 as positive FITC fluorescence (green) and tliat FITC fluorescence must be read only for nonfilamentous, 

 isolated cells in an area relatively free of clumps of fluorescent yolk sacs. 



114 



