in rabies - libdoc.who.int - World Health Organization

LABORATORY TECHNIQUES IN RABIES
Giycerol. Place the brain specimen In pure glycerol for 48 hours.
Phenol 0.5%. Make up 0.5O/' phenol in physiolog~cal salt solution, This is used
as the diluent for making up the 10% tissue inoculum. Hold for 6 hours or
overnight. If held for 6 hours, keep the suspension at room temperature. If held
for over 6 hours, keep the suspension overnight in the refrigerator.
Thiornersal' 1:5000. Make up a 1 :5000 solution of thiomersal in physiological
salt solution. This is used as the diluent for making up the 10% tissue inoculum.
Hold for 6 hours or overnight. If held for 6 hours, keep the suspension at room
temperature. If held for over 6 hours, keep the suspension overnight in the
refrigerator.
In order to determine whether contaminating bacteria are present, a portion of
all tissue emulsions should be cultured in dextrose iniusion broth or similar media,
and streaked on a blood-agar plate. The recommended amount is about 0.1 m1 of
emulsion in 3 ml of broth. The plate should be incubated overnight at 37.5"C.
Early deaths (1-3 days) among inoculated mice may be attributed to the presence
of contaminating bacteria if the cultures show moderate to heavy growth
and if many bacteria are found in the brain smears of the dead mice.
Annex
Preparation of Sellers' stain2
Examination of slide
In order to save time, the stained slide should rnitially be studied under low power
and areas containing numerous large neurons selected for examination under oil
immersion (Figs. 4.6 and 4.7).
Sellers' stain shows the Negri body well differentiated in magenta (heliotrope)
to bright red, with well-defined dark-blue to black basophlic inner bodies. All parts
of the nerve cell stain blue, and the interstitral tissue stains pink. Erythrocytes stain
copper red and can be easily differentiated from the magenta-tinged red of the
Negri bodies.
Stock solution
1. Methylene blue
Methanol (absolute acetone-free)
2. Basic fuchsin
Methanol (absolute acetone-free)
10 g
to make 1000 ml
5 9
to make 500 rnl
The stock soli~l~ons are stored in bottles with screw tops or ground glass
stoppers. Certified b~ological stains are preferable. The dry dyes should preferably
contain no less than 85% methylene blue, and no less than 92% basic fuchsin.
While absolute acetone-free methanol is recommended, chemically pure (C.P.)
methanol meeting American Chemical Society specifications may be substituted if
desired.
' Alsa known as th!merosai and rnercurothioiate
'This annex was klndly contrbuted by the late T. F. Sellers, Former Director Ernerrtus, Georgia Department
of Public Health Atlanta, GA USA.