GLANDERS 137 
(the anti-complementary action). A 1-10 dilution of the extract is first made by 
adding 1 ce. of the extract to 9 cc. of salt solution. From this 1-10 dilution other 
dilutions are prepared as follows: 
TABLE No. IV 
DILUTIONS OF THE ANTIGEN. 
No. | sdiition | Amount of Antigen | ie 
1 2 ec. lee. of 1-10 dil. 1-30 
2 |. 4ee. lee. of -110 dil. 1-50 
3 5 ce. lce. of 1-10 dil. 1-60 
4 7 ce. lec. of 1-10 dil. 1-80 
5 9 ce. lec. of 1-10 dil. 1-100 
6 lee. lec. of 1-100 dil. 1-200 
7 1% ce. 1 cc. of 1-100 dil. 1-250 
8 2 ce. 1ce. of 1-100 dil. 1-300 
9 3 ec. lec. of 1-100 dil. 1-400 
10 4 ce. lce. of 1-100 dil. 1-500 
The titration proper is carried out as follows. Eleven test tubes and three for con- 
trols are used. To each tube 1 cc. of salt solution is added, then 1 cc. of the complement 
of the previously determined smallest quantity established by the titration done before. 
To each tube add 1 ce. of the different dilutions (1-10, 1-30, 1-50, etc.) of the extract as 
obtained in Table IV. The tubes are shaken, placed in a rack and the rack placed in an 
incubator at 37° C. for one hour.* The tubes are then removed and 1 ce. of a double 
quantity of the previously titrated amboceptor is added to each tube, and finally 1 ce. 
of a 5% suspension of washed sheep blood corpuscles. Three control tubes are used. 
The first serves for a control to show that the complement alone (without the ambocep- 
tor) does not produce hemolysis, the second to show that the amboceptor alone does not 
produce hemolysis and the third that the salt alone does not produce hemolysis. In 
the first control tube 3 cc. of salt solution, 1 cc. of complement and 1 cc. of blood 
corpuscles are added. The second tube contains $ ce. of salt solution, 1 cc. of ambocep- 
tor and 1 cc. of blood corpuscles, while the third contains 4 cc. of salt solution and 1 cc. 
of suspension of sheep blood corpuscles. The following table (Table V) gives a sum- 
mary of the foregoing method. 
Each tube is shaken and all the tubes placed in the incubator at 37° C. for two 
hours.t Theresultsare then read. The tube in which hemolysis is no longer prevented 
represents the titre of the extract. In the glanders test, however, one-half that quan- 
tity is used. For example, if the results of the titration should be that the first tube 
*May place in water bath at 37° C. for ¥% hour. 
+Or water bath at 37° C. for one hour. 
