82 THE PRINCIPLES OF IMMUNOLOGY 



contamination. The suspension is pipetted into a sterile tube and the growth 

 killed by placing in a water bath of not less than 56 C. or more than 60 C. 

 for two hours. Rabbits are desirable animals because of the ease of intra- 

 venous injection. For ease in handling, the animal is placed in a box as shown 

 in Figs. 3 and 4. The ear is shaved along the course of the posterior auricular 

 vein situated near the posterior margin of the ear on its upper surface, is cleansed 

 with soap and water and sponged over with alcohol. Usually the alcohol makes the 

 vein stand out prominently, but if it does not, the ear may be pinched near its root 

 so as to distend the vein, or, if necessary, brushed over lightly with a sponge dipped 

 in xylol. Xylol should be used very sparingly, because of the danger of an 

 inflammation, which may make subsequent injections difficult. Bleeding from 

 the puncture may be stopped by pinching the ear for a few moments at the 

 site of injection. Usually one ear is used for injections and the other for 



PIG. 6. A flask placed upright after blood has clotted 



with oblique surface. The serum drains to the bottom 



of the flask and is easily withdrawn. 



test bleeding. The earlier injections or bleedings are near the tip of the ear, 

 the later ones approaching the base. The following protocol illustrates 

 an immunization: 



Day Killed typhoid emulsion 



I 0.05 agar slant * 



6 o.i agar slant 



ii 0.2 agar slant 



16 0.2 agar slant 



21 0.2 agar slant 



* If 10 c.c. saline had been added to 

 the culture, 0.5 c.c. suspension would 

 contain 0.05 agar slant. 



Preliminary Titration. One week after the last injection 0.5-1.0 c.c. blood 

 is withdrawn from an ear vein and the serum separated and titrated for the 

 agglutinin. (See Fig. 4.) If the titer is not satisfactory, the immunization 

 may be continued with living bacilli as follows : 



Day Living typhoid emulsion 

 I 0.05 agar slant 



4 o.i agar slant 



8 0.2 agar slant 



