302 IMMUNOLOGICAL METHODS OF DIAGNOSIS 



grams) of an eighteen-hour-old culture of the suspected organism, 

 suspended in 1 c.c. of broth. A second animal (B) is given ten titer 

 doses ( = 2 milligrams) with the same quantity of organisms. A 

 third (C) receives 50 multiples of the titer dose, i. e., 10 milli- 

 grams, of normal serum, but taken from an animal of the same 

 species as that furnishing the immune serum, together with the 

 same quantity of organisms as A and B, while a fourth guinea-pig 

 (D) is injected with the same dose of bacteria, but without any 

 serum. The animals should all be of about the same weight (250 

 grams), and are all injected intraperitoneally. To this end it is 

 recommended to make a small incision through the skin and to inject 

 through a cannula with a blunt point. By the aid of glass capillaries 

 a droplet of the peritoneal fluid is then procured through the same 

 incision, immediately after the injection, a second one twenty 

 minutes later, and a third one at the expiration of one hour. The 

 specimens are examined as hanging drops with an oil-immersion 

 lens. If the organism under consideration is the cholera vibrio, 

 typical granule formation and lysis will be observed in specimens 

 A and B after twenty minutes, and at the latest at the expira- 

 tion of one hour; while in C and D there will be large numbers of 

 actively motile organisms or such at least in which the form has 

 been w r ell preserved, the C animal being the control to A and B. 

 The object of injecting D is merely to prove that the organism in 

 question is virulent and this animal as well as C, of course, should 

 die, while A and B remain alive. If the result then turns out as 

 just indicated, the inference is justifiable that the organism under 

 examination was really the cholera vibrio. 



2. Pfeiffer's Test as Applied to the Recognition of Recent Cholera 

 Infections. In this case the individual's serum is diluted with broth 

 in the proportion of 1 to 20, 1 to 100, and 1 to 500, when guinea- 

 pigs are each inoculated as described above with 1 c.c. of various 

 dilutions, together with one oese (=2 milligrams) of an eighteen- 

 hour-old agar culture of a virulent cholera strain. If extensive 

 bacteriolysis can then be demonstrated at the expiration of twenty 

 minutes, or at most an hour, the inference is justifiable that the 

 person has recently passed through an attack of cholera. 



PREPARATION OF THE CHOLERA IMMUNE SERUM. The cholera 

 immune serum which is required in test 1 (above) is prepared as 

 follows: A number of rabbits are each injected intraperitoneally 



