140 GLANDERS 
numbered 1, 2, 3,and 4. One cc. of a physiological salt solution is added to tubes Nos. 
land3. Twoce. is placed in tubes 2and4. The serum of the suspected horse is then 
added. This serum has been previously rendered inactive, that is, the complement has 
been “inactivated” by heating for one-half hour at 56—-57° C. One-tenth cc. of this 
inactivated serum is added to tubes 1 and 2. Two-tenths cc. is added to tubes 3 and 
4, The antigen (glanders bacteria extract) is now added to tubes Nos. 1 and 3. One 
cc. of an established dilution is used. Tubes 2 and 4 are controls to see whether the 
suspected horse’s serum will influence hemolysis. 
To each tube is now added 1 cc. of a dilution of the complement (blood serum of 
normal guinea pigs). The proper dilution has been determined by titration as men- 
tioned before. 
A series of six controls should be made in connection with each series of tests carried 
out. One set of controls will do for a single day’s testing as long as the same substances 
(antigen, complement, amboceptor) are used in each test. It is also well to set what 
are called “positive” and “‘negative” controls in connection with each series of tests 
made. That is, the blood from a known glandered animal and likewise the blood from 
a healthy animal should be tested in connection with the blood from the suspected 
animals. 
The six controls mentioned above are made according to Table VI beginning with 
tube No. 5. 
Each tube is shaken carefully and placed in an incubator for 1 hour (or water bath at 
37° C. for 4% hour). This is done in order to allow the union or fixing of the comple- 
ment which will become locked up with the antigen and the bacteriolytic amboceptor 
in case the suspected serum came from a glandered animal. If the bacteriolytic ambo- 
ceptor is not present or the suspected serum was from a healthy animal the complement 
will not become locked up or fixed. 
After the required incubation period the tubes are removed from the incubator and 
to each tube is added 1 ce. of the previously titrated rabbit serum (hemolytic ambocep- 
tor). This serum has previously been inactivated by heating to 56°-57° C. for one-half 
hour, provided it has not been carbolized and kept for three days. Finally 1 ce. of a 
5% suspension of the washed red corpuscles of a sheep is added to each tube. The 
tubes are now replaced in the incubator and left for 10 hours, when the results may be 
read. If put in a water bath at 37° C. and left for 1 hour and then removed and kept 
at room temperature for from 3-5 hours the results may be read. Some workers prefer 
reading the results as soon as removed from the water bath. If the horse was affected 
with glanders, that is, the serum of the animal contained bacteriolytic amboceptor, no 
hemolysis will have taken place in tubes 1 and 3. The red corpuscles will have settled 
to the bottom and the upper liquid will be clear. The controls Nos. 2 and 4 should 
show complete hemolysis, that is, the fluid in the tubes should be uniformly red. If, 
however, the suspected serum came from a healthy horse and did not contain the 
bacteriolytic amboceptor, hemolysis should take place in tubes Nos. 1 and 2. Mohler 
gives the following advice in the interpretation of the results of the test: 
“Horses in which the serum produces a complete fixation of the complement in the 
quantities of 0.1 cc. and 0.2 cc. should be considered as glandered. 
“Horses in which the serum gives a complete fixation in the quantity of 0.2 cc. and 
an incomplete fixation in the quantity of 0.1 cc. should likewise be considered glan- 
dered. 
