126 PRACTICAL METHODS IN IMMUNITY. 



of the marginal veins of the rabbit's ear, and collect the blood in a 

 Wright's U-tube. Centrifugalizing, we have the serum ready for use. 

 The immune body and agglutinin in serum remain active for weeks 

 when kept in the refrigerator. The complement and opsonin, however, 

 begin to deteriorate at once and have disappeared by the fifth day. 

 Consequently, for opsonic and bacteriolytic and haemolytic experiments, 

 fresh serum 12 to 24 hours must be used, or it may be activated. 



AGGLUTINATION TESTS. 



There are two methods of testing the agglutinating powers of a 

 serum the microscopical and the macroscopical or sedimentation 

 method. 



i. For the microscopical method draw up serum to the mark .5 

 of the white pipette. Then draw up salt solution to the mark 1 1 . 

 This when mixed gives a dilution of i to 20. One loopful of the diluted 

 serum and one loopful of a bouillon culture or salt solution suspen- 

 sion of the organism to be tested gives a dilution of i to 40. One loop- 

 ful of the 1-20 diluted serum and 3 loopfuls of the bacterial suspension 

 give a dilution of 1-80. These two dilutions answer in ordinary diag- 

 nostic tests. The red pipette with a i-ioo or 1-200 dilution may be 

 used where dilutions approaching i-iooo are desired. Having mixed 

 the diluted serum and the bacterial suspension on a cover-glass, we 

 invert it over a vaselined concave slide and examine with a high 

 power, a dry objective (1/6 in.). It is simpler to make a ring of vaselin 

 to fit the cover-glass and make the mixture of diluted serum and culture 

 in the center of this ring or square. Then apply the cover-glass, press 

 it down on the vaselin ring and examine as with the ordinary hanging 

 drop. In making dilutions it is preferable to use salt solution, as the 

 phenomenon of agglutination requires the presence of salts. Ordi- 

 narily, 30 minutes is a sufficient time to wait before reporting the 

 absence of agglutination. Agglutination is more rapid at body tempera- 

 ture than at room temperature. In reporting agglutination, always 

 give time and dilution. It is absolutely necessary that a control prepa- 

 ration be prepared in every instance; that is, one with the bacterial 

 culture alone or with a normal serum of the same dilution as the lowest 

 used. Some normal sera will agglutinate in i to 10 dilution, and group 



