ACQUIRED IMMUNITY 69 



thirty minutes will suffice to kill them without too radically altering 

 the immunizing properties of the protein constituents. 29 



If the temperature is not raised above 60 C., and this is ad- 

 vised by many workers, the suspensions must be carefully controlled 

 by cultural tests before they are used, at least for the treatment of 

 human beings. As we shall see in a later section, the best results have 

 been obtained when heating was not carried beyond 53 to 55 C. 



When bacterial death is to be accomplished by chemicals the 

 antiseptics most commonly used are carbolic acid (0.5 per cent.), 

 toluol (removed before use of vaccine by filtration or evaporation), 

 chloroform, and formaldehyd (1 per cent.). 



Pfeiffer, who was one of the first to practice the immunization 

 of animals with dead bacteria on 'an extensive scale, believed that, 

 in the case of bacteria which were toxic by reason of their intra- 

 cellular constituents (endotoxins), the injection of the cell protein 

 itself, whether dead or alive, was the sole essential for successful 

 immunization. The method developed by Kolle 30 and by Pfeiffer 

 and Marx 31 for the prophylactic immunization of human beings 

 against cholera depends upon the injection of cholera cultures 

 emulsified in salt solution, killed by exposure to 58 C. for one hour, 

 and further insured against contamination by the addition of 0.5 

 per cent, phenol. The application of this method to other diseases, 

 both prophylactically and therapeutically, is more fully discussed 

 in another place. (See chapter XIX.) 



Since the essential point in such immunization is the introduc- 

 tion of the bacterial protein, it is often customary to inject bacterial 

 extracts instead of the whole cells. This has been especially desir- 

 able in the case of such insoluble micro-organisms as the tubercle 

 bacillus, where the injection of the whole dead organism produces 

 localized reactions similar to those caused by the living bacteria. 32 

 Thus a Old Tuberculin," as commonly used, is a glycerin-broth ex- 

 tract of tubercle bacilli. The method has been extensively used and 

 a variety of procedures have been devised for bacterial extraction. 

 These have included simple autolysis of the bacterial bodies in alka- 

 line broth, shaking in salt solution in mechanical shakers, trituration 

 with salt or sand, trituration after freezing, digestion with proteo- 

 lytic enzymes, and extraction by pressure in a Buchner press. 



We may mention some of the more important methods for pre- 



29 In a subsequent chapter (p. 258) we shall see that the physical changes 

 produced in an antigen by heat result in differences in the antibodies formed 

 after animal inoculation. This point has practical significance in the present 

 connection. See also the chapter on agglutinins, the work of Joos there dis- 

 cussed, and Friedberger and Moreschi, Centralbl. f. Bakt., 1905, Vol. 39. 



30 Kolle. Deutsche med. Woch., 1897, p. 4. 



31 Pfeiffer and Marx. Deutsche med. Woch., 1898. 



32 Prudden and Hodenpyl. N. Y. Med. Journal, 1891.* 



