134 GLANDERS 
The titration proper is then made in the following manner: Eight additional 
test tubes are each filled with 2.5 cc. of salt solution to which is then added the hemoly- 
tic serum (amboceptor) in quantities of 1 cc. of the different dilutions (Table No. I) to 
each tube. Afterwards the complement of the guinea pig serum is added in quantities 
of 0.5 ce. of a 10% dilution to each tube, and finally 1 cc. of a five per cent. suspension 
of washed sheep corpuscles in salt solution is placed in each tube. 
Besides these eight tubes there are also three control tubes, one to show that the 
complement alone will not produce hemolysis (without the amboceptor), the second 
that the amboceptor alone without the complement will not produce hemolysis, and 
the third that the salt solution alone will not produce hemolysis. Thus in the first 
control tube we add 3.5 cc. of salt solution, 0.5 cc. of complement, and 1 cc. suspension 
of sheep corpuscles. In the second control tube we add 8 ce. salt solution, 1 cc. of the 
1-100 dilution of the amboceptor and 1 cc. suspension of sheep corpuscles. In the 
third control tube we add 4 ce. salt solution and 1 cc. sheep corpuscles. A test tube 
rack with two rows of holes is very convenient for holding the tubes for these tests. 
TABLE No. II 
TITRATION OF RABBIT SERUM (HEMOLYTIC AMBOCEPTOR). 
Tube | (a) NaCl Amboceptor (b) Com | (c) Blood Remarks 
No. solution plement jcorpuscles 
1 2.5 ec. | 1 cc. of 1-10 dil. 0.5 ce. lee. 
2.5 ec. | 1ce. of 1-100 dil. | 0.5 ce. l ce. 
2 
3 2.5 ec. | lec. of 1-500 dil. | 0.5 ec. | Lee. 
4 
2.5 cc. | Lec. of 1-1000 dil.| 0.5 cc. lee. 
5 2.5 ec. | 1 ec. of 1-1500 dil. 0.5 ce. lee. 
6 2.5 cc. | 1ce. of 1- 2000 dil.} 0.5 ec. lee. 
7 2.5 ec. | lec. of 1-4000 dil.| 0.5 ce. | 1 ce. 
8 2.5 ce. | 1 ec. of 1-5000 dil.| 0.5 cc. | 1 ce. 
controls 
Complement control 
9 3.5 ce. 0.5 ce. | lce. {no hemolysis should occur) 
.. Amboceptor control (no 
10 3.0 ce. | 1 ec. of 1-100 dil. lee. hemolysis should occur) 
11 4.0 ce. lee. {Salt solution control (no 
hemolysis should occur) 
a. 0.9% NaCl solution. ‘ 
b. 0.5 cc. of a 10% solution of the ccmplement. 
c. 59% suspension of washed sheep corpuscles in salt solution. 
As can be seen from Table II the final volume in each tube is always uniformly 5 ce. 
Thus the different amounts of the blood derivatives used are always made up to.5 ce. 
by adding salt solution. 
