AGGLUTININS AND PRECIPITINS 



After 7-10 days a further titration is made, and if still unsatisfactory the 

 animal is discarded. As a rule, three animals are employed, and from these 

 at least one will produce an agglutinin which will titrate 1-5000 or higher. 

 The titer may be maintained by subsequent injections at longer intervals, but 

 it is usually found desirable to kill the animal by bleeding and to preserve the 

 serum in ampoules in the refrigerator. 



Bleeding the Immune Rabbit. The rabbit may be " bled out " by strapping 

 it on a flat board, lightly anesthetizing and plucking the hair from the groin 

 on one side. The skin is scrubbed 

 with soap and water and then 

 alcohol, and a long incision made 

 in the line of the groin groove. 

 (See Fig. 5.) This goes through 

 the f ascias and exposes the femoral 

 vessels. The neck of a sterile 

 150 c.c, flask is placed over the 

 vessels just below Poupart's liga- 

 ment and the vessels cut with the 

 knife, the blood being caught as 

 it spurts. As the bleeding con- 

 tinues the head end of the board 

 is raised and the animal's body 

 squeezed until the flow ceases. 

 Before disposing of the body, 

 death should be assured by a 

 blow fracturing the cervical spine. 

 The flask is placed in an oblique 

 position until the blood is firmly 

 clotted, then placed upright in 

 the refrigerator. (See Fig. 6.) 

 This leaves an oblique surface of 

 clot from which the serum flows 

 out in the bottom of the flask. 

 The blood may also be obtained 

 from the carotid artery, but this 

 requires more careful dissection, 

 longer anesthesia and may require 

 the insertion of a cannula. Practi- 

 cally it gives no better results 

 either in quantity of blood with- 

 drawn or sterility of the process. 

 Usually 15-20 c.c. serum are 

 obtained after twenty-four hours 

 in the ice-chest, and only occasion- 

 ally is it necessary to centrifuge in 

 order to obtain a clear serum. The 

 serum is withdrawn as shown in 

 Fig. 7 and placed in small sterile 

 ampoules of dark glass, sealed and 

 kept in the refrigerator. 



Macroscopic Titration. Titra- 

 tion is usually by the macroscopic 

 method, but an alternative is the 

 microscopic method. For the ti- 

 tration by the macroscopic method 

 it is necessary to have the growth 

 from two or three agar slants, 

 adding about 10.0 c.c. sterile salt 



solution to each tube and making an emulsion as described for immunization. 

 The suspension is placed in a flask and killed either by heat (56 C.-6o C. for 

 two hours), or by phenol i.o per cent, or formalin (40 per cent.) i.o per cent. 

 The killing of the organisms is not necessary, but is desirable because of the 

 added safety and because such killed emulsions may be preserved in the refrigera- 

 tor for several days or a few weeks. Broth cultures may be used, but the 

 hydrogen ion concentration of the broth may add a small factor of error not 

 present in the saline suspensions. 



PIG. 7. Method of drawing up measured volumes into 

 graduated pipette. The rubber tube enables the 



worker to observe the ascent of fluid in the pipette 



and the position of the tip of the pipette. Fluid is 



withdrawn from flasks in the same fashion. 



