BACTERIAL VACCINES . 2IQ 



provided prophylactic vaccination is instituted shortly after infection. One 

 can only determine whether or not an animal that has bitten a person has the 

 disease, by microscopic examination of the brain, and subdural inoculation of 

 rabbits with several drops of brain emulsion. 



When a suspected animal is killed for such an examination, injury of its 

 head should be avoided. Decapitation should be done so as to leave as much 

 as possible of the neck attached to the head. If the head must be held for some 

 hours or shipped before examination, it should be packed in a bucket of ice. 



The entire calvarium and posterior portion of the cervical vertebrae are sawed 

 away and removed without injury of underlying tissue before any attempt is 

 made to remove the brain and medulla. 



Films for Microscopic Examination are prepared as follows: An incision is 

 made at right angles to the surface and to the long axis of convolution into the 

 hippocampus major and cerebellum. Pieces of tissue about i millimeter thick 

 and several millimeters in diameter are removed, each placed near the end of a 

 glass slide and spread in a thin even film by sliding a second slide over the first 

 one while making gentle pressure. They are immediately immersed in methyl 

 alcohol for fixation. This must be done rapidly to prevent drying and conse- 

 quent tissue changes. After 3 minutes in alcohol slides are removed and stained 

 for 30 seconds with the following stain, freshly prepared. 



Engle's modification of Van Gieson's stain for negri bodies: 



Loeffler's alkaline methylene blue 5 cc. 



Distilled water 20 cc. 



Saturated alcoholic sol. f uchsin 4 drops. 



When properly stained the protoplasm of nerve cells is faint blue, nucleus purple 

 and nucleolus dark blue. If the blue is too intense the staining may be corrected 

 by adding more distilled water to the stain and heating slides while staining. 

 Negri bodies occur within the protoplasm of nerve cells, usually one, occasion- 

 ally several, appear in one nerve cell. They stain maroon red and contain one 

 or more deep purple or black inner bodies. 



Negri bodies, first described by A. Negri in 1903, are found in almost 99 per- 

 cent, of smears from brains of rabid animals, they have never been found in 

 other diseases; their nature is obscure. 



Negri bodies are round or oval and vary in size from i ju to 20 /z in diameter. 

 The smallest are observed in brain cells of rabbits inoculated with virus fixe; 

 the largest in the brain cells of cows afflicted with rabies. In the brains of dogs 

 dead from street infection they vary from barely visible to about 10 p. 



Inoculation of Rabbits. As soon as specimens have been removed from a 

 suspected brain for microscopic examination, the brain should be placed in 

 glycerin. If microscopic examination fails to disclose negri bodies, remove brain 

 from glycerin, excise from beneath the surface a piece of cerebellum about one- 

 half the size of a walnut, place in a sterile glass mortar with 15 or 20 cc. of sterile 

 normal salt solution and emulsify by trituration. Allow the suspension to stand 



