MICROBIAL diseases' OP MAN AND DOMESTIC ANIMALS 817 



37°. The sections adhere perfectly as a rule and are dry enough to proceed with 

 after ten to fifteen minutes. The sUdes are next transferred to xylol to remove 

 the paraffin and thence to absolute alcohol. The staining procedure is as follows: 



1. One minute in alcohohc eosin. 



Eosin extra B-Hoechst, 0'.$. 

 Alcohol, 60 per cent, loo.o. 



2. Wash in water. 



3. One minute in Loeffler's methylene blue. 



Saturated alcoholic solution of methylene blue, B-Patent Hoechst, 30.0. 

 Potassium hydroxide solution, o.oi per cent loo.o. 



4. Wash in water. 



5. One minute in Gram's solution. 



Iodine, i.o. 

 Potassium iodide, 2.0. 

 Distilled water, 300.0. 



6. Wash in water. 



7. Methylic alcohol until the preparation becomes entirely red. 



8. Wash in water. 



9. Loeffler's methylene blue again for thirty seconds. 



10. Wash in water. 



11. Dry carefully by pressing with filter paper upon the preparation. 



12. Differentiate in alkaline alcohol until only a weak eosin color remains in the 

 preparation. ' 



Absolute alcohol, 30.0 c.c. 



Sodium hydroxide, i per cent solution in absolute alcohol, 5 drops. 



13. Differentiate in acid alcohol until the collections of ganglion cells in the 

 gray matter are still faintly blue while the rest of the section is free from blue 

 (macroscopic). 



Absolute alcohol, 30.0 c.c. 

 Acetic acid, 50 per cent, i drop. 



14. Wash quickly in absolute alcohol. 



15. Xylol. 



16. Balsam and cover-glass. 



Steps 5 to 9, inclusive, may be omitted to save time at some sacrifice In the 

 final result. The Negri body is stained pink with blue granules in its interior. 

 The nerve cells are stained pale blue. 



Although sections are most satisfactory for diagnostic purposes and especially 

 to show the relation of the Negri bodies to the ganglion cells, it is usually possible 

 to recognize the Negri bodies in smears, after a little experinece. For this purpose 

 a portion of the gray matter of the Ammon's horn is crushed by gentle pressure 

 between two perfectly clean flamed slides and spread upon them by carefully slip- 

 ping the slides apart. The moist smears are at once fixed in methyl alcohol for 

 one minute, then washed in absolute ethyl alcohol, whereupon they are ready to be 

 stained by the procedure outlined above. 



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