514 HYDROPHOBIA 



causal agents, Grigorjew thinks such may be found in a protozoon which 

 he has constantly observed after inoculation in the cornea. 



In 1903 Negri described certain bodies as occurring in the 

 nervous system in animals dying of rabies to which considerable 

 attention has since been devoted, and regarding the significance 

 of which opinion is still divided. It may be said that Negri's 

 observations have been generally confirmed, and as it is probable, 

 whatever the final opinion as to the nature of the bodies 

 may be, that their occurrence is specific to the disease and hence 

 may be used for diagnosis, we shall describe the methods for 

 their demonstration. In doing so we shall chiefly follow the 

 work of the American observers, Williams and Lowden, who, 

 more than any others who have confirmed Negri, have used 

 methods widely employed in the investigation of similar 

 appearances. 



Their chief method is to take a piece of the brain tissue, to squeeze it 

 between a slide and cover-glass, and, sliding off the latter, to make a 

 smear which is then fixed in methyl alcohol for five minutes and stained 

 by Giemsa's stain (p. 107) for half an hour to three hours ; the prepara- 

 tion is then washed in tap water for 2-3 min. and dried. For rapid work, 

 after fixation, equal parts of distilled water and stain are used instead of 

 the more dilute mixture. 



For sections the tissues are left in Zenker's fluid l for 3-4 hours, then 

 placed in tap water for five minutes, 80 per cent alcohol with enough 

 iodine added to give it a port wine colour for 24 hours ; 95 per cent 

 alcohol and iodine, 24 hours ; absolute alcohol, 4-6 hours ; cleared with 

 cedar oil and embedded in paraffin of melting point 52 C. ; sections should 

 be 3-6 /u, thick. For staining, Mallory's methylene - blue eosin is 

 recommended ; the steps are as follows : xylol ; absolute alcohol ; 95 

 per cent alcohol and iodine, hour ; 95 per cent alcohol, ^ hour ; 

 absolute alcohol, hour ; eosin solution (5-10 per cent aqueous solution), 

 20 minutes ; rinse in tap water ; Unna's polychrome raethylene-blue 

 solution diluted 1-4 with distilled water, 15 minutes ; differentiation in 

 95 per cent alcohol for 1-5 minutes (the preparation being kept in 

 motion and its progress watched with a low power) ; rapid and careful 

 dehydration and clearing. 



The bodies vary much in size, measuring from - 5 //- to 25 p. 

 They are round, oval, or angular in outline. They are found in 

 the protoplasm of the nerve cells and of their processes. They 

 have a hyaline appearance with a sharply -defined outline, and in 

 their substance they contain granular material. Taking for granted 

 their cellular structure we may say that with the Giemsa mixture 



1 Zenker's fluid is of the following composition : potassium bichromate 

 2'5 gr., sodium sulphate 1 gr., perchloride of mercury 5 gr., glacial acetic 

 acid 5 c.c., water to 100 c.c. Dissolve the perchloride of mercury and the 

 bichromate of potassium in the water with the aid of heat and add the 

 acetic acid. 



