PFEIFFER'S REACTION 211 



of broth, and injected into the peritoneal cavity of a small guinea- 

 pig should cause granular degeneration and bacteriolysis of the 

 vibrios within one hour. 



Four mixtures are made (a) one loop of an eighteen -hour agar 

 culture of the vibrio to be tested, 0-001 c.c. cholera-immune 

 serum, suspended in 1 c.c. of broth ; (b) the same as (a), but 

 0-002 c.c. cholera serum ; (c) the same as (a), but 0-001 normal 

 serum of an animal of the same species as that furnishing the 

 cholera serum ; (d) one quarter loop of the vibrio in 1 c.c. of 

 broth, as a control of the virulence of the culture. These mixtures 

 are then injected into the peritoneal cavities of four guinea-pigs 

 each of about 250 grm. weight. At intervals of thirty and sixty 

 minutes hanging-drop preparations are made of the peritoneal 

 fluid of each animal, the fluid being obtained by inserting a capil- 

 lary pipette through a minute incision in the skin. In the guinea- 

 pigs injected with (a) and (6), if the organism be cholera, the 

 vibrios should show marked degenerative changes within sixty 

 minutes, while (c) and (d) will show plenty of active vibrios. If 

 the organism be non-virulent, two methods may be adopted for 

 applying the Pfeiffer reaction. The first, a microscopical or direct 

 method, is carried out by microscopical examination of hanging- 

 drop specimens of the organism suspended in a drop of the immune 

 serum to which a trace of fresh peritoneal fluid (complement) is 

 added. If the organism is homologous with the immune serum 

 the bacteria are soon transformed into granules. Controls are 

 put up at the same time with a known strain of the organism 

 with (1) its homologous immune serum + complement ; (2) non- 

 immune serum of the same animal + complement ; also of the 

 organism being tested with non-immune serum of the same 

 animal + complement. The peritoneal fluid may be obtained 

 by injecting 3-4 c.c. of broth into the peritoneal fluid of a guinea- 

 pig and four hours later withdrawing the fluid (now turbid with 

 leucocytes) and centrifuging, or allowing it to stand on ice for 

 twenty-four hours. 



In the second, or indirect, method, the organism is used to 

 prepare an immune serum by injecting an animal (e.g. a rabbit) 

 with it, and the immune serum so prepared is tested on a known 

 virulent strain in the peritoneal cavity of guinea-pigs in order to 

 ascertain whether or no it brings about bacteriolysis, i.e. the 

 Pfeiffer phenomenon. 



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