460 



METHODS AND FORMULAS 



DS 23.10 



out the red Rickettsial bodies. The modifi- 

 cation of Zinsser, Fitzpatrick, and Hsi 

 1938 (11189, 69:179) consists of differ- 

 entiating the magenta stain with citric 

 acid, and in staining in methylene blue 

 for a longer period; this, however, though 

 undoubtedly giving a better background 

 stain, does not give as clear a differentia- 

 tion of the Rickettsial bodies. 



Demonstration of Negri bodies in the 



brain of a guinea pig using the ethyl 



eosin-methylene blue technique 



of Stovall and Black 1940 



Negri bodies are usually diagnosed by 

 the smear technique which is described in 

 Chapter 8. The method here described is 

 intended more for the preparation of class- 

 demonstration material, though it may 

 also, when time is not vital, be used for 

 diagnostic purposes. The guinea pig has 

 been selected as a test animal, since it is 

 so commonly used for diagnostic infection 

 with the virus of rabies, the presence of 

 which in the brain causes Negri bodies to 

 appear in the cells, particularly those of 

 the horn of Amnion. It is presumed that 

 the investigator is acquainted with the 

 aseptic precautions which must be ob- 

 served in handling animals infected with 

 the virus of rabies. 



The guinea pig is killed, commonly with 

 chloroform, and tied down on its ventral 

 surface on a dissecting board. The head is 

 then skinned and the muscles dissected 

 away from the surface of the cranium. 

 Since only the anterior region of the brain 

 is required, it is advisable to cut with a 

 saw about yi inch anterior to the supra- 

 occipital bone vertically downwards for 

 about % of the distance through the skull 

 and brain. The supraoccipital and pos- 

 terior portions of the cranium are now re- 

 moved with a pair of powerful pliers which 

 will permit the cut end of the parietal 

 bone to be gripped and stripped off with- 

 out damage to the cerebral hemispheres. 

 It does not matter if the cerebral hemis- 

 pheres are shghtly damaged, since ample 

 protection will be afforded •to -the horn of 

 Amnion. The mcmlnanes of the brain 

 should be stripped off with forceps and the 



back of a large blunt knife inserted in the 

 slit between the two cerebral hemispheres. 

 A twist of the knife to the left will push a 

 hemisphere to one side, so that it can be 

 gripped firmly and drawn back until a 

 cartilage knife, or similar instrument, can 

 be used to sever the hemisphere from the 

 brain stem. This will leave the other cere- 

 bral hemisphere exposed so that it may 

 in its turn be severed. These two hemis- 

 pheres are now removed to a dish of 

 physiological saline, and the carcass of the 

 guinea pig destroyed with the customary 

 aseptic precautions. Each hemisphere is 

 now cut with a horizontal movement of a 

 large knife just about through the middle, 

 and the upper half is thrown away. This 

 will disclose the horn of Ammon at the 

 posterior end. A httle experience may be 

 necessary to determine the exact point at 

 which the cut should be made. Negri 

 bodies are most commonly found in the 

 basal portions of the horn of Ammon, and 

 from this portion a few pieces about ^i 

 centimeter in thickness and a centimeter 

 square should be removed to the selected 

 fixative. 



Stovall and Black (DS 23.13 Stovall 

 and Black 1940) suggest the use of their 

 method for diagnostic purposes by using 

 acetone as a fixative, which permits rapid 

 dehydration. For class-demonstration pur- 

 poses, however, it is better to use 5% 

 potassium dichromate in which the pieces 

 of brain are suspended in a loosely woven 

 cloth bag for a week or two to harden 

 them. 



If the sections are to be cut by an inex- 

 perienced technician, it is recommended 

 that every step of the procedure up to this 

 point be conducted by a physician, prefer- 

 ably a pathologist, and the pieces of brain 

 in potassium dichromate turned over to 

 the technician for further treatment. 



After the pieces have hardened for a 

 week or two, they are washed in running 

 water overnight, embedded in the cus- 

 tomary manner, and cut into sections of 

 about five microns in thickness. These 

 sections are mounted on the slide, de- 

 waxed in xylene, and run down through 

 the alcohols to distilled water. 



The great advantage of the technique 

 of Stovall and Black is that it involves the 



