160 
Dourine 
I. Preparation of Reagents. 
A. The Haemolytic System. 
[a) Red Cells. A quiet sheep may be bled in the standing position, 
otherwise it should be placed upon its back in a Y-shaped trough and 
held there by the attendant, an assistant shaving the neck and preparing 
the site of operation. The operator draws from the jugular vein, 
under aseptic conditions, 50 c.c. (more or less) of blood into a flask 
containing glass beads and in which the blood is defibrinated. It is 
then run through a double layer of fine, sterilized gauze into large centri¬ 
fuge cups, about 20 c.c. of blood in each, adding three to four times the 
amount of salt solution. The corpuscles are thrown down by centrifugal 
force, the upper fluid taken away and replaced with fresh salt solution, 
and the mixture again centrifuged. Washing in this way is repeated 
three times, when the red cells are carefully measured and suspended 
in an equal amount of salt solution, this 50 per cent, stock suspension 
being stored in the ice chamber until needed. 
(b) Haemolytic Serum. Rabbits have a variable amount of natural 
haemolytic amboceptor for sheep’s corpuscles—0-1 c.c. of fresh rabbit 
serum will usually haemolyze a like amount of 5 per cent, corpuscle 
suspension. For test purposes a serum with a much higher haemolytic 
index is required and to obtain this rabbits are hypersensitized or 
immunized by repeated injection of sheep’s corpuscles until a serum is 
given showing a haemolytic index of 0-0005. 
Not less than six large healthy rabbits should be selected for the 
immunization, for one or several are apt to die from shock during the 
process. The rabbits are injected intraperitoneally with a first dose 
of 2-5 c.c. of the 50 per cent, stock suspension of sheep’s corpuscles. 
Every 4-5 days a further injection is given, each time increasing the 
dose until, after five or six injections, it has reached 10 c.c. This dose 
is repeated once or twice. After the sixth or seventh injection 5-0 c.c. 
of blood is drawn from the heart of each rabbit, using a hypodermic 
syringe and a fine needle. The operation can easily be performed and 
does no harm to the animal. 
The serum of each rabbit is then heated for one half-hour at 56° C. 
and the haemolytic index established by titration (vide p. 164). It will 
be found, probably, that in only two or three rabbits out of six can the 
haemolytic index be raised to the desired degree, namely, 0-0005 or 
better. From such rabbits as much blood is drawn from the heart as 
