470 MICROBIOLOGY 



mined * and likewise 1 cc. of a 5% solution of sheep blood cor- 

 puscles. The amount of fluid in each tube is made up to 5 cc. 

 by the addition of salt solution. Three controls are used. The 

 first control tube contains 3.5 cc. 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 cc. salt solution, 1 cc. 

 of the amboceptor dilution and 1 cc. of the suspension of cor- 

 puscles ; in the third control 4 cc. of salt solution and 1 cc. 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 ex- 

 piration of this time they are removed and the results noted. 

 The highest dilution of complement in the tube in which the 

 hemolysis is complete 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 

 3, 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 cc. of salt solution. 



The amboceptor used in this titration should be inacti- 

 vated when fresh, or when preserved with chloroform. The 

 carbolized ambocepter may be used without inactivation after 

 it is 3 days old. 



4. Antigen. In testing for glanders this consists of an 

 extract of Bact. mallei prepared as follows. Cultures are made 

 on acid glycerine agar and allowed to incubate for 48-72 hours. 



* That is, if the titre was found to be 1-3000 by the previous 

 titration you would add 1 cc. of a 1-1500 dilution, as double the 

 quantity of amboceptor is added which has been stated before. 



* Here again the time may be shortened to one-half hour by the 

 use of a water bath at 37 C. 



