Indirect Immunofluorescence Test lor Legionnaires' Disease 



PREPARATION OF BACTERIAL ANTIGENS 



1 . Inoculate tlie organism lieavily on an agar slant of charcoal-yeast extract medium 

 (20- X 150-nim screw-capped test tube containing 10 ml of medium). Incubate the loosely 

 capped agar tube in a candle extinction jar at 35°C until good growth occurs (approxi- 

 mately 2 days). 



2. Add 2 ml of sterile distilled water to the tube. Gently rub the growth off the slant 

 with a Pasteur pipette, and transfer the suspension to a screw-capped test tube. 



3. Place the tube contaming the cell suspension in a boiling water bath for 15 min to kill 

 the cells. 



4. Streak an agar plate to confirm cell death. Use 0.1 ml of the cell suspension and the same 

 medium and incubation conditions as in Step 1. Observe for growth for at least 10 days. 



5. Pack the cells by centrifugation (2000 x g or about 5 min in a tabletop clinical centri- 

 fuge). Discard the supernatant fluid. Resuspend the bacterial sediment in 2 ml of sterile 

 distilled water (concentrated antigen suspension). 



6. Make working dilutions of the concentrated antigen suspension in 0.5'^^ normal chicken 

 yolk sacs (NYS) in sterile distilled water. (The procedure for preparation of NYS is de- 

 tailed in the manual Appendix.) it is generally best to try several antigen dilutions in the 

 1:20 to 1:80 dilution range in the indirect IF test. High concentrations of bacterial cells 

 can reduce titers, because proportionately fewer antibody molecules are bound per bac- 

 terial cell, and low concentrations make slide reading difficult because there are too few 

 fields of cells and too few cells per field. Most antigens are dispersed optimally at a dilution 

 of 1:50 (OD of 0.08-0.10 at 660 nm with 1-cm light path or approximately 500-600 organ- 

 isms per microscopic field at a magnification of 3 15 X). 



7. Add the preservative, sodium azide (NaN3 ), to the concentrated antigen and to the 

 diluted "working" antigen to a final concentration of 0.05%. 



8. Store antigens at 4°C. Although it is too early to know the results of long-term stability 

 studies, antigens are apparently stable at 4°C for at least 2 mon. 



INDIRECT IMMUNOFLUORESCENCE TEST PROCEDURE 



A. Preparation of antigen slides 



1 . Thoroughly mix the working dilution of the heat-killed antigen suspension. Using a 

 Pasteur pipette, apply enough antigen to each well of a microscope slide to cover the well 

 with the antigen; remove the excess liquid with the same pipette (e.g., 25- X 75-mm acetone- 

 resistant glass slide with 12 staggered wells 5 mm in diameter; Cel-Line Associates, Inc., 

 Minotola.N.J.). 



2. Allow the slide to air dry (may take up to 30 min). 



3. Fix the antigen smears to the slide by placing the slide in an acetone bath for 15 min. 



4. Allow the slide to air dry. 



5. If slide is to be stored for future use, place in freezer at -20°C. Frozen slides are stable 

 for at least 2 mon. 



B. Titration of Sera 



1. Prepare a 1:16 dilution of the serum in 2.5%-3.0% normal chicken yolk sac (NYS) 



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