168 
Dourine 
Mix well and incubate for one hour and ten minutes at 38-39° C. 
Mix together equal quantities of haemolytic serum (amboceptor) 
and red cell suspension, then add 1-0 c.c. of the mixture to each tube. 
Shake again and incubate for two hours longer. 
It is usually possible to read the antigen titre in 1| hours and proceed 
with the final tests; nevertheless, the tubes should be left or replaced 
in the incubator for the full two hours and then put on one side for 
further reference and to see if any further action has taken place. 
Tube No. 10 is the control for the haemolytic system and must 
show complete haemolysis. No. 10 in the second set contains only 
haemolytic amboceptor and red cells and must not show the slightest 
degree of haemolysis. Nos. 8 control the horse serum, Nos. 9 the antigen, 
the red cells being haemolysed in all. 
The positive set will show more or less complete fixation of comple¬ 
ment—no haemolysis, except perhaps in the first and second tubes, 
the negative set complete haemolysis. When the antigen appears 
very strong there may be some inhibition in the negative set in the tubes 
containing the larger amounts of antigen. 
The amount of antigen to be selected as the titre for the final tests 
is that which gives complete fixation with the positive serum while 
double the quantity in the corresponding tube of the negative set does 
not prevent or inhibit haemolysis. 
III. The Serum to be tested. 
Collection of Serum. The chief point aimed at in collecting blood 
from suspected animals is sterility, especially when the specimens have 
to be transported over long distances and mailed to the laboratory. 
Absolute sterility is not essential, nevertheless as near as possible 
aseptic conditions are to be strongly recommended and the avoidance 
of adding carbolic acid or any other antiseptic fluid to the sample speci¬ 
men as a preservative. The blood clot should be well formed and the 
serum odourless and clear or only slightly tinged with haemoglobin. 
The condition of a sample of blood may vary greatly according to 
the size and shape of the vial or tube containing it, the slowness or 
rapidity with which blood is run into the vial, the partial or complete 
filling of the vial, the shaking of the specimen before coagulation has 
occurred, and in other ways irrespective of aseptic conditions and of 
abnormal properties of the blood itself. In square or rectangular 
bottles and in specimen vials without a neck the clot has a tendency 
