136 GLANDERS 
to produce complete hemolysis in the presence of one amboceptor unit and a suspension 
of sheep corpuscles.* The serum from each guinea pig should be titrated in the follow- 
ing manner before it is used. A basic dilution of 1-10 is made of the complement by 
adding 0.3 cc. of the complement to 2.7 cc. of salt solution in a test tube. 
From this dilution certain definite amounts are added to each of five other tubes 
according to Table III. To the complement thus distributed to each tube, is added 
1 ce. of hemolytic amboceptor of which the titre has already been determined{ and 
likewise 1 cc. of a 5% solution of sheep blood corpuscles. The amount of fluid in each 
tube is made up to 5 ce. by the addition of salt solution. Three controls are used. 
The first control tube contains 3.5 ce. salt solution, 0.5 cc. of the 10% basic dilution of 
complement and 1 cc. of a suspension of sheep corpuscles. In the second control 3 ce. 
salt solution, 1 cc. of the amboceptor dilution and 1 cc. of the suspension of corpuscles; 
in the third control 4 cc. of salt solution and 1 ce. of the suspension of sheep’s corpuscles 
are used. The order of adding each product and the amount of each are given in 
Table III. 
After shaking each tube, they are placed in a rack and then in an incubator at 37° C. 
for two hours.{ After the expiration of this time they are removed and the results 
noted. The highest dilution of complement in the tube in which the hemolysis is com- 
plete indicates the titre of the complement. For example, if hemolysis is complete in 
the tube where 0.3 cc. of the 10% basic dilution of the complement was used (Tube 
8, Table III) and the hemolysis is incomplete in the tube in which 0.2 cc. of the same 
dilution was employed (Tube 4, Table III) then the titre of the complement is 0.3 cc., 
inasmuch as a 10% basic dilution of the complement was employed. Thus, in the 
tests using this complement it would be necessary to employ a 3% complement dilution, 
that is, 3 cc. of the serum of the guinea pig in 97 ce. of salt solution. 
The amboceptor used in this titration should be inactivated when fresh, or when 
preserved with chloroform. The carbolized amboceptor may be used without inactiva- 
tion after it is 3 days old. 
Antigen. In testing for glanders this consists of an extract of Bact. mallet prepared 
as follows. Cultures are made on acid glycerin agar and allowed to incubate for 48-72 
hours. The growth is then washed off with salt solution. This salt solution suspension 
of the organisms is kept at 60° C. for four hours in order to kill the bacteria. The sus- 
pension is then placed in a shaking machine and shook at frequent intervals for four 
days. It is then centrifuged at high speed (8000-5000 revolutions per minute), the 
clear supernatant liquid drawn off and ten per cent. of a 5% solution of carbolic acid 
added. Before use the antigen must be titrated to obtain its anti-complementary 
action. It would be well also to determine its antigenic and hemolytic properties as 
well. However, this may be left until the test proper. 
Titration of the extract or antigen. The titration of the extract is carried out in 
order to determine the quantity of the extract which no longer prevents hemolysis 
*The serum is obtained by bleeding a strong vigorous guinea pig by cutting the 
throat. The pig is first anesthetized and the ventral portions of the neck shaved and 
disinfected. The blood is collected preferably in a Petri dish. 
{That is, if the titre was found to be 1-3000 by the previous titration you would add 
1 ce. of a 1-1500 dilution, as double the quantity of amboceptor is added which has been 
stated befcre. 
ice again the time may be shortened to one-half hour by the use of a water bath 
at 87°C. 
