Detection ol Lcpionnnircs; Disease Baeteiia in Clinical Specimens by Direct Ininiunotltiorescence 



2. Stain all preparations by covering the smear nearest the labeled end of the slide with 1-2 

 drops (0.05 ml) of the preimnuine or other control conjugate. 



3. Cover the second smear with 1-2 drops of the use dilution of the polyvalent LDB conjugate. 



4. Place the slides in a covered chamber to prevent evaporation during staining. 



5. Stain for 20 min. 



6. Remove excess conjugate by holding the slide level, but perpendicular, and tapping it against 

 a towel. Quickly and gently rinse smears with a stream of PBS from a wash bottle while keeping 

 the slide rougjily horizontal with the long edge tipped slightly downward. Prevent the specific 

 conjugate from coming into contact, even momentarily, with the control smears. 



7. Immerse slide in PBS for 5 min. Use a separate PBS container for each slide to prevent 

 cross-contamination with organisms that may wash off of LDB positive slides. 



8. Rinse each slide in distilled water from a wash bottle. 



9. Air dry. 



10. Add a small drop of buffered glycerol (pH 9.0) and a 1 or 1'/: (Coming) cover sHp to the 

 smears. 



EXAMINATION OF STAINED SLIDES 



Examine first under the lOX objective of the tluorescence microscope. In strongly positive 

 preparations the bacteria may be visible as unifonnly sized dots. Select areas of the smear where 

 organisms may be present, and switch to a 40X or 63X oil immersion objective for screening. Use 

 the lOOX oil objective for close study and confinnation. The bacteria will be visible as single 

 short rods or small intracellular or extracellular clumps of organisms showing strong peripheral 

 staining with darker centers. No specimen should be reported as negative until after at least a 

 5 -min search. 



If organisms of appropriate morphology are stained by the polyvalent LDB conjugate and 

 not be the control conjugate, stain the additional smears with the LDB serogroup-specific re- 

 agents. 



CONTROLS 



The most important controls consist either of preimmune conjugate from the same animals 

 which furnished the specific reagent or conjugates prepared from the sera of unimnumized 

 animals of the same species as those immunized. These control reagents should have approxi- 

 mately the same protein content and F/P ratio as the specific conjugate. 



As controls, the conjugates should be used at either the same dilution as that of the specific 

 conjugate, or preferably, at twice that concentration, as a margin of safety. 



Each specimen preparation showing specific staining must be tested with the control reagent 

 to insure that the observed reaction is serologically specific. Other essential controls are culture 

 suspensions, and positive and negative preparations of tissue scrapings, tissue imprints, sections, 

 and smears of lower respiratory tract secretions, depending upon the types of diagnostic material 

 being submitted for examination. 



Positive and negative smears should be carried through the specimen staining procedure each 

 time a group of specimens is processed. Negative smears may be made from suspensions of any 

 heterologous bacteria which are not related serologically to the LDB. 



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