MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 871 



Transverse sections, 2 to 3 mm. in thickness, of the Ammon's horn of the sus- 

 pected brain are hardened in acetone at 37 for one hour, then transferred to 

 melted paraffin (melting point 55) in the paraffin oven at 58 for one and one-half 

 hours and embedded. Sections, 2 to 3ju in thickness, are then cut with the micro- 

 tome, floated upon lukewarm water and mounted upon perfectly clean flamed 

 glass slides. The excess of water is carefully removed with filter paper and the 

 slides are then completely dried on a warm plate at 45 or in the incubator at 

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

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

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



1. One minute hi alcoholic eosin. 



Eosin extra B-Hoechst, 0.5. 

 Alcohol, 60 per cent, 100.0. 



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 100.0. 



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. 



n. 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. 



1 6. 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. 



