Detection ol' Legionnaires' Disease Bacteria in Clinical Specimens by Direct Immunonuorescence 



PREPARATION OF SPECIMEN MATERIAL 

 Tissue scrapings of Formalin-fixed tissue (■/) 



1. Select one or more areas of the lung or other tissue for testing. With hing tissue, choose dense 

 areas of gray or reddish consolidation. 



2. Transfer each tissue block to a sterile petri dish. 



3. With a sharp scalpel, cut through these areas to produce new tissue faces for scraping. 



4. Grasp the tissue with forceps, and holding the scalpel at a right angle to the tissue face, scrape 

 it to produce a fine puree of tissue particles. The lung tissue of victims of Legionnaires' disease is 

 usually quite friable. If the tissue is rubbery or spongy, a positive test is unlikely. 



5. Using the scalpel blade, smear the particles of tissue and tissue tluids onto two 1.5-cm circles 

 on each of three microscope slides. Stain one smear on the first slide with preimmune or normal 

 conjugate and the other with the polyvalent conjugate. Reserve the other four smears for staining 

 with the four monovalent serogroup conjugates if the polyvalent conjugate gives positive results. 



6. Let the smears air dry. Gently heat fix. It is not necessary to remove the Formalin before 

 staining. 



Fresh or fresh-frozen tissue (process in a safety cabinet) 



When fresh or fresh-frozen tissue is obtained through autopsy or biopsy, the first laboratory 

 procedure should be to inoculate culture media for attempts to isolate the LDB. This will 

 minimize contamination of the tissue. Then select tissue for other tests, including direct FA. 



1. Using sterile instruments, cut a fresh face of tissue; with the forceps, press and squeeze the 

 tissue against three clean microscope slides, making impression smears within the two 1.5-cm 

 circles on each slide. If the tissue is so moist that smears may be too thick, blot on sterile gauze 

 or culture media before imprints are made. 



Alternatively, grind the tissue with sterile phosphate buffered saline (PBS) and alundum in a 

 sterile mortar, or homogenize the tissue in a Ten Broeck or comparable tissue grinder. Use 

 sufficient PBS to give an approximate 10% tissue homogenate. Prepare smears in the same 

 manner as described for Formalin-fixed tissue. With larger pieces of tissue this method produces a 

 more representative sample and reduces sampling error. 



2. Air dry and heat fix. 



3. Fix smears for 10 min by covering them with 10% neutral Formalin. Do not let the Formalin 

 dry on the smears. 



4. Drain off the Formalin and gently and briefly dip each slide into distilled water to remove the 

 remainder of the Formalin. (If slide is not rinsed, Fomialin will not interfere with staining.) 



5. Air dry. 



Tissue sections 



Legionnaires' disease bacteria maintain their serologic integrity through histopathological 

 processing and can easily be demonstrated in tissue sections if they are present in reasonable 

 numbers. However, they are not as easily demonstrated in such sections as they are in scrapings 

 of Fomialin-fixed lung or imprints of fresh lung tissue because many of the bacteria are intra- 

 cellular, lie at many different levels in the section, and are shrunken by the histological process- 

 ing. Counterstains, such as rhodamine-labeled rabbit serum or rhodamine-labeled bovine serum 

 albumin, make the organisms much more visible in tissue sections. (See Addendum at the end of 



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