Oct. 15,1917 Conglutination Test for the Diagnosis of Glanders 
67 
TECHNIC OF THE CONGLUTINATION TEST 
Five factors enter into the conglutination reaction as in complement 
fixation: (1) Serum to be tested, (2) antigen, (3) complement (horse 
serum), (4) inactivated cow serum (conglutinin), and (5) sheep blood 
corpuscles. 
A negative reaction is indicated by conglutination (clumping) of the 
red blood cells, the corpuscles being spread out in the bottom of the 
tube with a distinct ragged border. On shaking the tube the large 
clumps of red blood cells are very easily seen. Some hemolysis may take 
place, in which case the fluid is stained. In a positive reaction no con¬ 
glutination takes place, the* blood cells settling to the bottom of the 
tube in a mass, piling one on top of the other, with a very sharp regular 
outline, leaving an absolutely clear fluid above. The corpuscles settle 
to the bottom of the tube more slowly than in a negative case, as in the 
negative case the corpuscles are clumped which makes them settle 
more rapidly than the individual cells. On shaking the tube in a positive 
(+) case the blood corpuscles leave the bottom of the tube with a charac¬ 
teristic whirling, the fluid assumes its original opacity, and no clumping 
of the corpuscles is observed. 
Certain differences in the technic of the conglutination test exist 
among the several investigators, the principal ones being (A) to add all 
the ingredients at once in the following order: suspected serum, antigen, 
horse complement, inactivated cow serum, and sheep blood, to allow 
the tubes to stand at room temperature, and to read the results in two to 
four hours; or (B) to add suspected serums, antigen, and complement, 
incubating for one hour at 37 0 C., then to add the cow serum and sheep- 
blood corpuscles, returning the ingredients to the incubator and reading 
the results from in four to nine hours. The technic employed in the 
work of the writer is similar to that employed by Pfeiler and Weber (9), 
with some few changes, and consists in combining all the reagents one 
after the other, allowing them to stand at room temperature, and 
reading the results in two hours. 
The best results were obtained by following this technic: 
Preparation and titration of conglutination system— 
1. Fresh normal horse serum (complement). 
2. Inactivated cow serum (conglutinin). 
3. Washed sheep-blood corpuscles. 
1. Horse serum, or complement. —A sufficient quantity of blood 
is drawn from the jugular vein of a normal horse. The blood is allowed 
to clot and after a time the serum is poured off. It is important that 
the serum be absolutely clear and free from red cells. The horse comple¬ 
ment should be used fresh—that is, the same day on which it is drawn— 
as it can not be used after 24 hours. 
