THE VIRUS OF HYDROPHOBIA 615 



minutes and stained by Giemsa's stain (p. 116) for half an hour to three 

 hours ; the preparation is then washed in tap water for 2-3 minutes 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 : 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 Jiours j cleared with 

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

 be 3 to 6 M 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 methylene-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. 



Frothingham recommends a method of making "impression prepara- 

 tions" of the brain. The part (e.g., hippocampus) is laid on a piece of 

 wood whose porosity causes it to adhere ; a clean slide is then lowered 

 upon the tissue and slight pressure applied ; on raising the slide a 

 thin film of cells preserving their original arrangement is lifted off, 

 and this can be fixed and stained like a smear, van Gieson's method 

 being used by this author. 



We have found that the bodies can be perfectly well demonstrated 

 by fixing the brain in formalin, preparing paraffin sections and staining 

 by Irishman's method. 



The Negri bodies (Plate IV., Fig. 16) 2 vary much in size, 

 measuring from '5 to 25/i,. They are round, oval, or angular 

 in outline. They are found in the protoplasm of the nerve cells 

 and of their processes. When examined in unstained prepara- 

 tions, they are seen to have a sharply denned outline, and some 

 of the features of the internal structure presently to be described 

 can be noted. With regard to staining reactions, they are 

 frankly eosinophil for certain combinations containing eosin, 

 e.g., alcoholic eosin-methylene-blue, Mann's eosin mixture, and, in 

 certain circumstances, Leishman's stain. For the finer differen- 

 tiation of the internal structure, Negri employed Giemsa's stain. 

 With this stain and under high magnification the groundwork 

 of the body is a pale blue ; in it there appear certain round or 

 oval, multiple or single formations, of varying size, stained pink, 



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. 



2 For the material from which this preparation was made we are indebted to 

 Capt. W. F. Harvey, I.M.S. 



