74 METHODS FOR EFFECTING ACTIVE IMMUNIZATION OF ANIMALS 



preparing the emulsion. This difficulty may be reduced to a minimum 

 by thoroughly washing the organ or tissue prior to preparing an emulsion 

 for injection. 



The following meth'od, after Pearce, illustrates the mode of preparing 

 a nephrotoxic serum by immunizing rabbits with dog kidney. 



1. Anesthetize a dog with ether, open the abdomen, wash the blood 

 out of the kidneys by inserting a cannula high up in the abdominal aorta, 

 and flushing with from six to ten liters of salt solution; open the vena 

 cava. 



2. Remove the kidney as aseptically as possible, and grind it in a 

 meat-grinder; rub through fine-meshed wire gauze, and wash the residue 

 in several changes of sterile salt solution. The fat should be rejected 

 and only the cortex used. 



3. Weigh and suspend 10 grams of the substance in 30 c.c. of sterile 

 salt solution. 



4. Inject the rabbit intraperitoneally with 10 c.c. of the emulsion 

 every seven days. 



5. After the third dose has been given test the serum, as subsequent 

 doses increase the danger of losing the animal. 



6. The animal is bled one week after receiving the last injection. 



7. The injection of this serum into dogs is usually followed by al- 

 buminuria and possibly hemoglobinuria. This subject is further con- 

 sidered under the head of Practical Exercises with Cytotoxins. 



Some investigators have asserted that by effecting immunization 

 with the nucleoproteids of an organ more specific cytotoxic serums are 

 secured. These claims have not been confirmed by Pearce, Wells, 

 and others. (See Chapter XXV.) 



Nucleoproteins may be secured as follows: Grind the organ or tissue in a meat- 

 grinder, and finally rub up with sand with a pestle in a mortar; add two volumes of 

 normal salt solution, and pass through a meat press; collect the effluent, place in a 

 refrigerator for twenty-four hours and then filter through gauze and centrifuge the 

 filtrate; to the supernatant fluid add acetic acid to remove the nucleoproteins. Place 

 in the refrigerator for eighteen hours and centrifuge. Collect the sediment and wash 

 several times with normal salt solution. Dissolve the sediment in normal salt 

 solution containing 0.5 per cent, sodium carbonate. Reprecipitate with acetic acid, 

 wash, and redissolve in the alkaline solution. 



