METHODS OF SERO-DIAGNOSIS. 20< ) 



solution being used. To this mixture we further add in each 

 tube i cc of a 5 % suspension of red corpuscles of the sheep in 

 physiological salt solution. Control tubes to show that neither 

 the rabbit serum, nor the guinea-pig serum, nor the salt solution, 

 is by itself capable of producing haemolysis, are also employed. 

 Finally, the volume of all the tubes is brought up to uniform 

 standard by the addition of physiological salt solution, and the 

 tubes, after shaking, are placed for two hours in an incubator 

 at 37° C. The smallest quantity of the rabbit serum producing 

 complete haemolysis of the corpuscles at the end of this time 

 indicates the titer of the serum and, for the proper performance 

 of the test, this should be very low. We use twice this quantity 

 in applying the test. 



Next it is necessary to find what is the smallest amount of 

 the complement used which is capable of producing the complete 

 haemolysis of the standard amount of sheep corpuscles in the 

 presence of twice the smallest quantity of haemolytic rabbit serum 

 which caused complete haemolysis of the corpuscles in the last 

 test. The complement, as before-mentioned, is obtained by using - 

 guinea-pig serum, and this must be used relatively soon after it 

 is obtained since its activity gradually decreases on keeping and 

 ultimately disappears. The titration is carried out on the same 

 principle as before by taking a series of test tubes, to each of 

 which is added I cc of a solution containing the previously deter- 

 mined necessary amount of rabbit serum, then I cc of 5 % sus- 

 pension of sheep's corpuscles, then the complement in varying 

 quantities, and finally the volume of fluid in the tubes is made 

 uniform by the addition of physiological salt solution. The 

 same control tubes are used as in the previous test. All of these 

 tubes are shaken, placed in the incubator at 37 ° C for two hours 

 and then examined. In this case the tube which shows complete 

 haemolysis of the contained corpuscles indicates the smallest 

 quantity of complement which, in the presence of the determined 

 amount of haemolytic rabbit serum, is capable of producing com- 

 plete haemolysis of 1 cc of a 5 % suspension of sheep's red cor- 

 puscles in physiological salt solution inside two hours at 37 ° C. 

 The antigen is an extract of the glanders bacillus (B. mallei) 

 made from a young culture which has been- grown on some solid 

 medium, such as glycerin-potato-agar. The extract is made in 

 physiological salt solution, to which has been added a small 

 quantity (.5 %) of carbolic acid as a germicide. In the case of 

 the antigen we must also determine by titration what is the most 

 suitable quantity to use, since we know that if present in too 

 large quantities it may directly combine with some of the com- 

 plement ; hence we must find the largest amount which will not 

 interfere with the production of complete haemolysis by the 

 quantities of complement and haemolytic serum before deter- 

 mined, and, in applying the test, we use half of this amount of 

 antigen. 



The titration of the antigen is based on the same principles 



as the previous titration tests, the quantities of complement, has- 



molytic rabbit serum, and blood, present in the tubes being con^r"\ P a 



/\, OS Ay 



