68 METHODS FOR EFFECTING ACTIVE IMMUNIZATION OF ANIMALS 



3. With soluble alien proteins, as serum, milk, egg-albumen, etc., 

 as in the manufacture of precipitins. 



4. With various alien cells, as erythrocytes, kidney cells, sperma- 

 tozoa, etc., in the manufacture of cytotoxic serums, such as hemolysins, 

 nephrotoxin, spermatotoxin, etc. 



PRODUCTION OF ANTITOXIN 



Diphtheria and tetanus antitoxins are prepared on a large scale by 

 the inoculation of horses with increasing doses of the respective toxins. 



The methods for preparing these antitoxins, and also of antimeningo- 

 coccus, antistreptococcus, antipneumococcus, and other curative se- 

 rums are given in the chapter on Antitoxins and Passive Immunization. 



PRODUCTION OF AGGLUTININS 



Agglutinating serums are frequently of much value in making a 

 bacteriologic diagnosis of typhoid fever and cholera. For this purpose 

 unknown microorganisms are mixed with proper dilutions of known 

 immune serum, and the presence or the absence of agglutination noted. 

 As a rule, rabbits are used in the preparation of these serums; for the 

 production of larger quantities of serum, goats and horses are occasionally 

 employed. 



The injections may be given intravenously or intraperitoneally; 

 occasionally the first injections are given subcutaneously, to be followed 

 later by intraperitoneal injections. By heating the cultures at a temper- 

 ature not exceeding 60 C. for from one-half to one hour, there is less 

 danger in the subsequent handling of the cultures and agglutinins are 

 readily produced. 



1. Use forty-eight-hour agar cultures of the organism, such as 

 Bacillus typhosus, Spirillum cholerae, etc. Bouillon cultures may be 

 used, but are not recommended on account of the various other con- 

 stituents present in the medium. 



2. With a sterilized three-millimeter platinum loop remove one 

 loopful of culture and rub up in 2 c.c. of sterile salt solution in a small 

 test-tube until a homogeneous emulsion is secured. 



3. Heat the emulsion for thirty minutes at 60 C. in a water-bath. 



4. Inject intravenously. 



5. Give four more injections at intervals of a week as follows: 



Second dose: 2 loopfuls in 2 c.c. NaCl solution, heated. 

 Third dose: 4 loopfuls in 2 c.c. NaCl solution, heated. 

 Fourth dose: 6 loopfuls in 2 c.c. NaCl solution, heated. 

 Fifth dose: 1 agar slant in 4 c.c. NaCl solution, heated. 



