AGf~ICULTURAL RESEARCH, PUSA.

210 PRA.CTICAL BACTRRlOLOGY
HAEMOLYTIC SYSTEM
With guinea - pig complement a haemolytic system
consisting of ox or sheep .red corpuscles, sensitised
with the appropriate immune serum is used.
Defibrinated blood is obtained at thc abattoir (vide
p. 102) and washed thrcc 0]' four times with normal
8aline, being centrifugalised after each washing to
separate the corpm;cles. The centrifuged deposIt of
corpuscles after thc final washing is suspcndcd in normal
saline to f01'm n 3 PCl' cent. suspension, and ftvc
minimum haemolytic doses (M.H.D.) or the immune
serum arc added.
Burroughs Wellcome haemolytic serum for sheep red
corpuscles may conveniently be used in preparing a
haemolytic system for the test. Anti-sheep haemolytic
sera tend to exert a pronounced agglutinating effect
on the homologous corpuscles with rapid sedimentation
of the cells; we prefer, therefore, to use for
complement - fixation tests ox corpuscles with an
anti-ox hacltlolysin obtained from thc rabhit.
'l'he mcthod of prcparing a HAEMOLYTIC ANTISEIWlIl and
of estimating its M.H.D. may be summarised as follows :a
rabbit is injected intravenously at weekly intcrvals with
increasing fitllUIlllts of washcd rcel cclls-c.g. 0'5 C.c., 1'0 c.c.,
1'5 C.c. of the sediment aftcr washing and centrifugalisatioll.
A week nfter the last injection, a small quantity of hlood is
withdrawn from an ear vcin; thc serum is separated, and its
M.ll.D. cstimated by testing the haemolytic effect of varying
amounts (c.g. from 0'001 c.c. to 0'005 c.c.) on 1 C.c. of a a per
cent. sllspension of rcd cells along with fill exccss of guinca-pig
complemcnt (e.g. 0'05-0'075 c.c.) (vide methods given below).
As guinea-pig serum contains natural huemolysins for ox
fill(1 sheep corpusclcs, the complement-containing scrum must
previously be frced from this antibody by mixing cqual
volumes of thc guinca-pig scrnm and the appropriate red
corpuscles (washed), keeping the mixturc at 0° C. for onc hour
and thcn sepamting the scrum by centrifuging. If the M.ll.D.
is 0'001. c.c.-0·002 c.c. the animal is bled from thc neck vcsscls
or by cardiac IHmcture (virZe p. 1(7). 'rhe blood scrum is separated,
heated for onc hoUl' at 55° C., and storcd in sealed quill
tubes (vide p. 20:3).