18
always be recognised with certainty after the tissue has been soaked in glycerine two or more
days. We failed after this procedure to recognise the bodies on some occasions though the inocu-
lation was positive, but succeeded on others.
Tissue fixed in alcohol 90 per cent. or formalin 5 per cent. will enable the bodies to be
recognised provided that a volume of fluid, at least 20 times the bulk of the piece, be used, but
one cannot rely on good preparations with either of these fixatives.
A rapid diagnosis can generally be made by teasing out a fragment of a fixed hippocampus
in water, when Negri bodies can be clearly made out in the isolated and unstained nerve cells.
We have not relied on this performance for a positive diagnosis, much less for a negative one,
but it has so far been supported by the finds in the stained sections.
To demonstrate Negri bodies for certain the best all round stain seems to be Mann's
watery eosin and methyl blue mixture. The bodies show up fairly well after staining with any
acid dye and differentiating, but for routine use Mann's stain has certain advantages over
them all. It is not necessary to adhere rigidly to the instructions given by Mann, two hours'
immersion in the stain with subsequent differentiation is ample, though a stay of 24 hours if
convenient does no harm.
A more rapid stain is that of Unna which gives beautiful pictures and more structural
detail than Mann's, but it is not so reliable for mere diagnosis.
Rosanilin violet is at times a useful stain when no contrast colour is required. Half an
hour in a saturated aqueous solution and differentiation in 90 per cent. alcohol depicts Negri
bodies very clearly but without much detail. Some experience of it is desirable, otherwise
errors may be fallen into.
The internal structure of the bodies is best brought out by various manipulations with
alcoholic eosin and alkaline methylene blue followed by differentiation in acid and alkaline
alcohols, which cannot be repeated with invariable success, far less described. Instead of
paraffin sections the smear and dab methods described by several writers should be of value
when dealing with fresh, unfixed brains. Teasing out fresh hippocampus in dilute acetic acid
does not often display very much. The large nerve cells are so delicate and their processes are
so interlaced that they can seldom be freed from their connections in the fresh state. Such a
preparation shows little but cell nuclei, which are tougher than the cytoplasm, and myelin
globules.
Dabs and smears—if whole, uninjured, isolated nerve cells can. be rapidly fixed on the slide
— o u g h t
t o
p r o v i d e a m e a n s o f s t u d y i n g t h e i n c l u d e d N e g r i b o d i e s s o m e t h i n g b e t t e r t h a n s e c t i o n s ;
but, owing to the delicacy and entanglement of the cells alluded to above, it is exceedingly
difficult to get preparations worth anything at all.
In order to establish whether we could reasonably expect to find Negri bodies in the brains
of animals sent for examination, which had been killed because they had been behaving in a
"peculiar" way and might have been in the early stage of rabies, the brains of six rabbits
were examined which had been inoculated subdurally with a street virus which in the previous
passage had had an incubation period of 13 days.
TABLE No. 10.
Rabbit
number. num
Days after
inoculation.
Symptoms.
Microscopic findings.
1
7
Nil.
Nil.
2
9
Nil.
Small Negri bodies in hippocampus; commencing changes in Pur-
kinjé's cells, but no bodies; degeneration of cells of spinal ganglia,
but no bodies.
3
10
Nil.
Hippocampus, bodies small and scanty; cerebellum, small bodies,
numerous; cervical spinal ganglia, bodies in some cells, degenera-
tion of others.
4
11
Commencing
paralysis.
Hippocampus and cerebellum, bodies numerous and of medium size;
cervical spinal ganglia, plentiful.
5
12
Marked paraly-
sis.
Hippocampus, cerebellum, ganglia, contained numerous bodies large
and small.
6
Died, not rabies.
Table 10 shows that in all probability at least two days before any outward and visible sign
of rabies appears Negri bodies are sufficiently large and numerous to be readily observed in the
hippocampus, and that 24 hours before there are outward signs Negri bodies are to be found in
all parts of the brain and. also in the cervical spinal ganglia.