170 BACTERIOLOGICAL METHODS 



or from serum institutes or laboratories. Horse serum is not as 

 toxic to rabbits as are some other sera. Meat extracts should 

 always be filtered to avoid infection of the animals to be im- 

 munized, but extensive sloughing is likely to occur with any 

 method of immunization and the mortality rate is high. The 

 blood or serum used as antigen can be preserved by the addition 

 of chloroform (1-2 per cent.), or by drying. 



On account of the individual differences existing in rabbits 

 in regard to the development of precipitins, it is advisable to 

 treat at least six animals at the same time. The injections of 

 2-3 cc. of horse or deer serum are made at intervals of 5 days. 

 Ten days after the last injection the blood is tested for pre- 

 cipitins. The further treatment of the animals differs individu- 

 ally, depending on the precipitin contents of the rabbits. Ani- 

 mals which show a high precipitin reaction are given subsequent 

 inoculations subcutaneously or intraperitoneally, to avoid ana- 

 phylactic death which frequently results from intravenous in- 

 oculations. Some rabbits fail to produce precipitins, whatever 

 the method used. 



Fornet and Mulled recommend the intraperitoneal injection 

 of 5, 10 and 15 cc., respectively, of protein material on the ist, 

 2d and 3d day, respectively. The test for antibodies is carried out 

 on the 1 2th day. Gay and Fitzgerald 1 inject on three consecutive 

 days i cc. of the antigen, bleed, and test the serum on the icth 

 day. Both methods frequently give very good results. The 

 precipitin content of an immune serum is occasionally titrated 

 during the process of immunization by withdrawing a few cubic 

 centimeters of blood from an ear vein. The hair over the marginal 

 vein is removed and the skin rubbed with alcohol. A fine pipette is 

 introduced into the vein and the blood collected by capillary attrac- 

 tion or by suction. It is, however, advisable for the beginner to 

 cut the vein transversely and to collect the blood in a centrifugal 

 tube. The hemorrhage is stopped by covering the wound with 



1 University of California publications, Pathology, Vol. II, 75, 1912. 



