Demonstration of the Bacterium in Tissue by a Modification 

 of the Dieterle Silver Impregnation Stain 



A.E. Van Ordeii and P.W. Greer 



The modification of the Dieterle silver impregnation stain described below is our adaptation 

 of the staining procedure which Dr. Robert Dieterle developed for demonstrating Spiruchaeta 

 pallida in single microscopic sections. We modified his procedure to meet our need for a batch 

 staining method and have used it successfully for a number of years. Recently, interest in this 

 modified method increased after we used it successfully to demonstrate the Legionnaires' disease 

 bacterium (LDB) in paraffin-embedded tissue sections. The routine staining procedures for bac- 

 teria and fungi were not satisfactory; therefore, the modified Dieterle staining procedure is a 

 valuable diagnostic tool. Instructions for the procedure are detailed below. 



TISSUE AND SLIDE PREPARATION 



Fix lung tissue for at least 24 hours in 20 times its volume of \Q7r buffered Formalin. 

 Trim the tissue with a sharp blade to not more than 3 x 1.5 \ 0.5 cm. Process according to tlie 

 following schedule: 



1. Place in 70% alcohol (two changes of 1 hour each). 



2. Transfer to 957c alcohol (two changes of 1 hour each). 



3. Transfer to absolute alcohol (two changes of 1 iiour each). 



4. Place in equal parts of absolute alcoiiol and chloroform for 1 hour. 



5. Transfer to chloroform (two changes of 1 liour each). 



6. Place in melted paraffin (two changes of 2 hours each). 



7. Place tissues in a third change of melted paraffin until tiiey are embedded. 



8. Transfer tissue to an embedding mold and fill the mold with fresh melted paraffin. 

 Allow the paraffin to harden, remove the paraffin block from the mold, and trim its face of 

 excess paraffin. 



9. Cut thin sections (4-6 micrometers) from the paraffin-embedded tissue blocks and 

 float the sections on warm water in a floatation water bath. If a pinch of gelatin was dis- 

 solved in the warm water, pick up a floating tissue section with a plain glass slide. When 

 gelatin was not used, pre-coat the glass slides with egg albumin before picking up sections. 

 (NOTE: Albumin on the slide surface may be a source of nonspecifically staining artifacts.) 



STAINING PROCEDURE 



Glass or plastic staining racks must be used or the staining can be done in coplin jars. Metal 

 containers must not be used because the staining reagents react witii metals to form a black pre- 

 cipitate which adheres to the tissue sections. All glassware must be thoroughly cleaned. Include 

 a known positive control in each staining rack or coplin jar. 



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