Detection of Legionnaires' Disease Bacteria in Clinical Specimens b\ Direcl Imniuiuilliiorescence 



this chapter for counterstain preparation.) Hvans" blue or otiier counterstains will also probably 

 be helpful, but their relative value has not been sufficiently documented. 



1 . Cut tissue sections as thin as possible (4 fjni or less) from paraffin blocks. 



2. Affi.x the sections to slides by heatinu them for appro.ximately 15 min at 58° to (iO°C. 



3. Remove the paraffin and hydrate the tissue sections by sequentially dipping the slides in two 

 changes each of xylene, absolute ethancil, 95'^? ethanol, and distilleti water. 



4. Air dry. 



Lung exudates and pleural fluids (process in a safety cabinet) ( /, 6. iV ) 



Expectorated sputa, transtracheal aspirates (TTA), bronchial washings, pleural fluids, and 

 other specimens from the lower respiratory tract (LRT) are all satisfactory materials for study. 

 Patients with Legionnaires' disease (LD) frequently do not produce much sputum, and what they 

 produce may not be purulent. The secretions are usually extremely viscid and tenacious. Smears 

 usually contain tissue cells which will autofluoresce or stain nonspecifically, so counterstains are 

 needed. Until selective media are developed, it is probably not worthwhile to culture expec- 

 torated sputum; however, clean specimens such as TTA, bronchial washings, and pleural tluids 

 that are taken by invasive procedures should be cultured. Several isolates have been obtained in 

 this way. 



1. Select a viscous portion of the sputum, aspirate, washing, or other LRT specimen, and 

 prepare two smears of moderate thickness on each of three microscope slides. Pleural tluids tend 

 to form a fibrin clot on the slide, and unless processed carefully, the entire film can be dislodged. 



2. Air dry and heat fix. 



3. Fix smears in \07o neutral Formalin for 10 min. Either place each slide in a separate coplin jar 

 or lay each one flat and flood it, but do not let the Fonnalin dry on the slide. 



4. Drain off the Formalin. 



5. Airdi-y. 



Blood cultures 



Inoculate 5 ml of patient's blood into a bottle of charcoal yeast extract (CYE) diphasic 

 blood culture medium (see chapter by Feeley et al., this manual). Incubate at 35°C aerobically. 

 Each day flood the agar slope of the diphasic medium with- the bnUh portion. Growth may not 

 be apparent for four or more days. When growth is apparent on the agar medium, subculture to 

 slants of CYE agar and Feeley-Gonnan (F-G) agar. Prepare smears for direct FA, Gram's and 

 other stains by standard and previously described methods. 



Culture smears (process in a safety cabinet) 



1. Make suspensions of known or suspected LDB cultures in 1',^ Fomialm m 0.85/f NaCl 

 solution to give a light turbidity (McFarland No. 2). 



2. Prepare smears on double-ring slides. 



3. Air di7 and heat fix. 



FA STAINING PROCEDURE 



1. Methods for preparing the specific LDB conjugate and a counterstain reagent are detailed in 

 the Addendum to this chapter. 



94 



