54 LABORATORY COURSE IN SERUM STUDY 



twentieth of the emulsion obtained from an agar slant, and after ten 

 minutes a specimen of the peritoneal fluid is withdrawn for observation. 

 This is done by shaving the left side of the abdomen of the pig, holding 

 the pig, back down, on a tray. This can be done satisfactorily with the 

 operator's left hand. The shaved surface of the skin is sterilized by 

 wiping with 5 per cent carbolic, a small cut made through the skin, 

 but not through the muscles, with a sharp pair of scissors. Then the 

 sharp tip of a capillary pipette is inserted through the muscles and the 

 peritoneum. If the pipette is held horizontally or with the open end 

 depressed, a small amount of peritoneal fluid will run up into the capillary 

 portion even if no suction is used. When sufficient amount of fluid 

 [0.1 or 0.2 c.c.] is obtained, the pipette is withdrawn and the puncture 

 covered with cotton soaked in carbolic solution. 



A hang-drop preparation is made with a drop of this fluid and the 

 remainder blown out into a watch glass. With a platinum loop a series 

 of thin smears are made of this fluid on glass slides, allowing one slide 

 for each member of the class, and as soon as the films are dried they are 

 placed in Coplin jars containing a 10 per cent solution of formalin 

 (4 per cent formaldehyde) for five minutes. They are then withdrawn, 

 allowed to dry, and are stained by the students with methylene blue and 

 studied under the oil immersion lens. The hang-drop preparation 

 should be observed under a high-power dry lens and one preparation 

 may be observed by the whole class. 



Preparations are made in this way from the normal and immune pig 

 ten minutes, thirty minutes, one hour and two hours respectively after 

 injection. By the end of this period if a proper dose of the organisms 

 has been injected, the spirilla will be found to have multiplied enor- 

 mously in the peritoneal fluid of the normal animal, whereas in the 

 smears made from the immune animal after thirty minutes to an hour 

 the spirilla will appear granular or swollen, and after two hours, or, if 

 the observation is followed that long, after three or four hours, will be 

 found to have practically disappeared. 



The two animals should be kept for observation after the experiment, 

 and the unprotected animal will probably be found dead the next day. 



The passive transference of bacteriolytic immunity to another 

 animal by simultaneous injection of cholera spirilla and anti-cholera 

 serum obtained from the rabbit immunized to cholera may be demon- 

 strated in a similar way, and by this technic the strength of a bacteriolytic 

 serum may be titrated by injecting series of guinea pigs with varying 

 amounts of the serum. 



