Demonstration of the Bacterium in Tissue by a Modification of the Dieterle Silver Impregnation Stain 



1 . Remove the paraffin and hydrate the tissue sections by sequentially dipping the slides 

 in two changes each of xylene, absolute alcohol, 95% alcohol, and distilled water. 



2. Place slides in preheated 5% alcohohc uranyl nitrate at 55°- 58°C for 1 hour. 



3. Dip slides in distilled water. 



4. Dip slides in 95% alcohol. 



5. Place slides in 10% alcoholic gum mastic for 3 minutes. 



6. Dip slides quickly in 95% alcohol. 



7. Place slides in distilled water for 1 minute. 



8. Allow slides to drain for 15-20 minutes. (Slides can be left to dry overnight and the 

 procedure completed the following day.) 



9. Place slides in preheated 1% silver nitrate solution in 55°- 58°C oven in the dark for 

 at least 4 hours. (The silver will precipitate if the oven temperature exceeds 60°C.) 



10. Dip slides twice quickly in distilled water. 



1 1 . Dip sHdes in developing solution until sections are yellow to tan-usually in 1-2 minutes 

 (check control slide microscopically for proper development of organisms). 



12. Dip slides twice in distilled water. 



13. Dip slides twice in 95% alcohol. 



14. Dip slides twice in acetone. 



15. Dip slides twice in xylene. 



16. Place shdes in a second dish of xylene. 



1 7. Coverslip as soon as possible with Permount or Protex mounting medium. 



INTERPRETATION 



Scan the sections on a microscope with the low power objective. Locate areas of consolida- 

 tion in which the alveoli are filled with fibrin and leukocytes. In most cases, we have found more 

 organisms in the field containing tlie most degenerated leukocytes. 



Under the high dry objective, the LDB appears as a 0.4-0.8 micrometer by 2-4 micrometer 

 dark-brown to black rod against a yellow to tan background. Although these organisms may be 

 intracellular or extracellular, most are found within macrophages. Spirochetes and some other 

 bacteria also stain dark-brown to black. In paraffin-embedded tissue sections, bacteria which 

 stain dark-brown to black with this procedure and have the morpiiological characteristics of the 

 LDB but do not stain with tissue Gram stains can be presumed to be the LDB (Figure 1 ). 



Other structures including melanin granules, chromatin, Formalin pigment, and some 

 foreign material in macrophages may also stain by this method, but they can be readily distin- 

 guished from tiie LDB. 



DISCUSSION 



The modified Dieterie silver impregnation stain has also been successfully applied to de- 

 stained sections previously stained by hematoxylin and eosin or by tissue Gram stains. After 

 removing the coverslips (by soaking slides in xylol) and destaining the sections in acid alcohol 

 (by dipping until colorless), we have laed the modified Dieterle procedure to demonstrate LDB 

 in sections tiiat have been filed for as long as one year. 



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