GLANDERS 135 
After adding the substances to the test tubes in the order given, the tubes are well 
shaken, placed in the test tube rack, and put in an incubator at 37° C. for two hours. 
Then the tubes are removed from the incubator and the results read.* 
The highest dilution in which complete hemolysis has taken place represents the 
titre of the hemolytic amboceptor. Thus if complete hemolysis has taken place up to 
and including the tube where the dilution of the rabbit serum was 1-2000 (Tube No. 6, 
Table ITI), the hemolytic titre of this serum is represented by 1-2000. This dilution, 
however, is not used in the glanders test; but rather its double strength, which would 
be 1-1000. The titre of the hemolytic amboceptor for use in the diagnosis of glanders 
should not be less than 1-1000 and therefore if the rabbit serum should prove to be of a 
lower titre it should be discarded. It often happens that some rabbits are found to be 
unsuited for the production of amboceptor and the titre of their serum can not be 
raised high enough even after repeated inoculations with washed sheep corpuscles. 
It is advisable to preserve the hemolytic amboceptor in small vials containing 1 to 2 
ce. of the rabbit serum, and seal the corks with paraffin or sealing wax. 
Complement. The complement which is contained in the blood serum of a normal 
guinea pig is employed. The complement should be titrated to determine its efficiency 
to act with the hemolytic amboceptor. 
Titration of complement. By titration of the complement there is aimed to be 
established a complement unit which is the smallest quantity of complement necessary 
TABLE No. III 
TITRATION OF COMPLEMENT. 
Complement 
Tub NaCl x, Amboeep- 
No. | edlanoe (a) gone © tor (c) Blood corpuscles (d) 
1 2.5 ce. 0.5 ce. lee. lee. 
2 2.6 ce. 0.4 ce. lee. lee. 
3 2.7 ec. 0.3 ce. 1 ee. lee. 
4 2.8 cc. 0.2 ce. lee. 1 ee. 
5 2.9 cc. 0.1 cc. lee. l ce. 
controls 
6 3.5 ce. 0.5 ce. 1 cc. complement control 
(no hemolysis should occur) 
7 3.0 ce. lee. | 1 ec. amboceptor control (no hemolysis 
should occur) 
8 4.0 cc. 1 ce. salt solution control (no hemolysis 
shculd occur) 
a. 0.9% NaCl solution. as 
b. Guinea pig serum in diminishing quantities. __ . : 
ce. Of previously titrated hemolytic serum, double dissolving quantity. 
d. 5% suspension of washed sheep blood corpuscles in salt solution. 
*If the tubes are placed in a water bath kept at 37° C. the time here may be shortened 
to one-half hour. 
