IMMUNITY 411 



commence the process with non-fatal doses of cultures killed by 

 the vapour of chloroform or by heat, the doses being gradually 

 increased, and afterwards to proceed with increasing doses of the 

 living organism. In this way a high degree of immunity against 

 the organism is developed, and further, the blood serum of an 

 animal thus immunised (anti-cholera serum) has markedly pro- 

 tective power when injected, even in a small quantity, into a 

 guinea-pig along with five or ten times the fatal dose of 

 the living organism. Under these circumstances the spirilla 

 undergo a granular transformation and, ultimately, solution ; this 

 phenomenon is generally known as Pfeiffer's reaction, and was 

 applied by him to distinguish the cholera spirillum from organisms 

 resembling it. The following are the details : 



Pfeiffer's Reaction. A loopful (2 rngrm.) of recent agar culture of the 

 organism to be tested is added to 1 c.c. of ordinary bouillon containing 

 '001 c.c. of anti-cholera serum. The mixture is then injected into the 

 peritoneal cavity of a young guinea-pig (about 200 grm. in weight), and 

 the peritoneal fluid of this animal (conveniently obtained by means of 

 capillary glass tubes inserted into the peritoneum) is examined micro- 

 scopically after a few minutes. If the spirilla injected have been cholera 

 spirilla, it will be found that they become motionless, swell up into 

 globules, and ultimately break down and disappear positive result. If 

 they are found active and motile, then the possibility of their being 

 true cholera spirilla may be excluded negative result. In the former 

 case (positive result) there is, however, still the possibility that the, 

 organism is devoid of pathogenic properties and has been destroyed by 

 the normal peritoneal fluid. A control experiment should accordingly 

 be made with *001 c.c. of normal serum in place of the anti-cholera serum. 

 If no alteration of the organism occurs with its use, then it is to be 

 concluded that a true reaction has been given. 



The serum of an animal immunised by the above method has 

 also marked agglutinative action against the cholera spirillum, 

 and this property closely corresponds with Pfeiffer's reaction as 

 regards specificity. Such a serum has, however, little protective 

 effect against the toxic action of the dead spirilla. On the other 

 hand, Macfadyen by injecting the endotoxin derived from the 

 spirilla by grinding obtained a serum which had antitoxic as well 

 as agglutinative and bacteriolytic properties (vide Immunity). 

 Metchnikoff and others have also obtained antitoxic sera which 

 act on the extra-cellular toxins obtained by filtration. 



The serum of cholera convalescents has been found to possess 

 properties similar to those of immunised animals ; that is, it 

 affords protection against the cholera spirillum and may also 

 give Pfeiffer's reaction. These properties of the serum may be 

 present eight or ten days after the attack of the disease, but 

 are most marked four weeks after ; they then gradually become 



