Detection of Legionnaires' Disease Bacteria in Clinical Specimens by Direct Immunofluorescence 



2. Counterstain (rhodamiiie-labeled rabbit serum) 



Deparaffinized tissue sections and sputum smears are difficult to examine for LDB by 

 tiie direct FA test because of the brigiit background formed by tlie nonspecific uptake 

 of fluorescein-labeled globulin. Whole nomial rabbit serum labeled with tetramethyl- 

 rhodamine isothiocyanate has been found to be useful as a counterstain with these 

 types of specimens. 



a. Materials 



1. Whole serum from nonimmunized rabbits. 



2. Tetramethylrhodamine isothiocyanate (TMRD. This reagent can be purchased 

 in 100-mg lots from Baltimore Biological Laboratories, Baltimore, Maryland, or 

 from Research Organics, Inc., Cleveland, Ohio. 



3. Phosphate buffered saline (PBS) - 0.5 M sodium phosphate, pH 7.6, contain- 

 ing 0.85% NaCl and 0.1% NaNj . 



4. Dim ethyl form amide (DMF). 



5. 0.5MNa3PO4. 



6. Sephade.x G-25 fine (Phamiacia Fine Chemicals, Piscataway, New Jersey). 



b. Procedures 



A 40-ml aliquot of whole serum is adjusted to pH "5.5 by adding 0.5 M Na,, PO4 . A 

 100-mg portion of TMRI is dissolved in 2 ml of DMF before being mixed with 

 serum. The reaction mixture is allowed to stand for at least 1 h at room tempera- 

 ture and then centrifuged to remove particulate matter. Unreacted rhodamine 

 products are removed by gel filtration on a Sephadex column. A column 5 cm in 

 diameter by 30 cm in length packed with 100 g of Sephadex G-25 fine that has 

 been presoaked in PBS is adequate for this purpose. The first colored fraction to 

 emerge from the column is the rhodamine-labeled serum. This fraction is followed 

 by an orange fraction and a red fraction which are discarded. The column can be 

 washed fairly clean of rhodamine products and used repeatedly. The whole 

 serum -rhodamine conjugate should be dialyzed against PBS to remove traces of 

 DMF, adjusted to a 160-ml volume by adding PBS, and filtered through a 0.45 ^m 

 fOter. 



c. Application 



The anti-LDB conjugate, at four times the usual working concentration, is com- 

 bined with three parts of the counterstain. This mixture is used in the usual 

 manner for FA staining of LDB in tissue sections or in sputum specimens. If the 

 anti-LDB conjugate is in the lyophilized state, it is rehydrated by adding the 

 appropriate volume of counterstain to produce the desired working concen- 

 tration. When a counterstain is used with the diagnostic conjugate, a similarly 

 prepared control conjugate must also be used. 



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