GLANDERS GROUP 257 







minutes before it can be used. Fresh guinea-pig serum is used as 

 complement. The antigen used is an extract of glanders bacilli 

 prepared from the growth on slant glycerin-agar tubes. The 

 growth is washed off with physiologic salt solution and heated to 

 60 for four hours to kill the bacteria. The suspension of organ- 

 isms is then placed in flasks and shaken in a shaking apparatus for 

 four days. It is then centrifuged, the clear liquid removed, and 

 10 per cent, of a 5 per cent, solution of phenol added. This anti- 

 gen may be preserved without material deterioration for several 

 months if kept in a cool, dark place. 



It is necessary to titrate the rabbit serum and likewise the 

 antigen in order to determine the amounts most suitable for carry- 

 ing out the test. For each set of determinations of diagnosis fresh 

 guinea-pig serum must be used. Blood-serum from the animal 

 that is suspected of having glanders must be inactivated by heat- 

 ing to 58 for thirty minutes. The materials necessary for the 

 test are 



1. Washed sheep corpuscles, 5 per cent, suspension (antigen 1). 



2. Inactivated serum from rabbit immunized against 1 (am- 

 boceptor 1). 



3. Fresh guinea-pig serum (complement). 



4. Extract of glanders bacilli (antigen 2). 



5. Inactivated serum from suspected animal (amboceptor 2). 

 The test is carried out in test-tubes. In tubes 1 and 2 there is 



placed 0.1 c.c. of the serum (No. 5, above), and in tubes 3 and 4, 

 0.2 c.c. of the same. One c.c. of the established dilution of glanders 

 bacilli (No. 4, above) is then added to tubes 1 and 3. To each tube 

 is then added 1 c.c. of the dilution of fresh guinea-pig serum that 

 has been established by preliminary test. Each tube is now made 

 up to 3 c.c. with physiologic salt solution. They are then placed 

 in the thermostat at 37 for an hour. They are then removed and 

 to each tube is added 1 c.c. of the previously standardized rabbit 

 serum (No. 2, above) and 1 c.c. of the sheep corpuscles (No. 1, 

 above). The tubes are shaken and incubated for ten hours. 

 A positive diagnosis is indicated by lack of hemolysis in tubes 1 and 

 3 and complete hemolysis in tubes 2 and 4. Checks must be made 

 to determine the hemolytic activity of each of the above con- 

 stituents independently. 



17 



