128 LABORATORY BACTERIOLOGY 
(3) The diagnosis by the presence of Negri bodies. These 
bodies may be found in a microscopic examination of properly 
stained preparations made in either one of three ways ;. namely, 
. (a) Sections of the affected brain properly fixed and stained. 
(2) Impression preparations of the Ammon’s horn or cerebellum. 
(¢) Smears. 
The technic of the smear method is as follows : 
(1) A small bit of the gray substance of the brain chosen 
for examination is cut off with a sharp pair of scissors and placed 
about one inch from the end of a glass slidé so as to leave 
room for a label. The cut in the brain should be made at right 
angles to the surface. 
(2) Another glass slide is placed over the piece of tissue and 
pressed upon it until it is spread out in a moderately thin layer, 
then this latter slide is moved slowly and evenly acrdss to the 
end opposite the label. Only slight pressure should be used in 
making the smear, but slightly more should be exerted on the 
slide toward the label end, thus allowing more of the nerve 
tissue to be carried further down the smear and producing more 
well-spread nerve cells. If any thick places are left at the edge 
of the smear they should be spread out toward the side of the 
slide. 
(3) Such a smear should be made from at least three differ- 
ent parts of the brain. First, from the Ammon’s horn ; second, 
from the cerebellum ; third, from the cortex in the region of 
the crucial fissure. 
(4) These smears are fixed in heat, preferably over a mild 
flame from a Bunsen burner. Care must be taken not to heat 
too much. 
(5) The smears are then stained for from 10 to 15 minutes 
in a saturated solution of alcoholic eosin. This stain works 
better if it is at least 3 months old. 
(6) Remove smear and wash in tap water. Dry carefully 
between filter paper and stain for from 10 to 30 seconds with 
Loeffler’s alkaline methylene blue. 
