TECHNIC OF BACTERIOLYTIC TESTS 367 



4. Mix 2 c.c. of the third dilution (1 : 500) with 2 c.c. of bouillon 

 (= 1 : 1000). Place 2 c.c. in a separate tube and add 2 loopfuls of cul- 

 ture. Inject 1 c.c. ( = 0.001 c.c. immune serum). 



5. To 1 c.c. of the fourth dilution (1 : 1000) add 9 c.c. of bouillon 

 (=1 : 10,000). Place 2 c.c. in a separate tube, and add 2 loopfuls of 

 culture. Inject 1 c.c. ( = 0.0001 c.c. immune serum). 



6. Control: Emulsify 2 loopfuls of culture in 2 c.c. of bouillon. In- 

 ject 1 c.c. This animal will probably succumb within twenty-four hours. 



7. Control: To 2 c.c. of a 1 : 10 dilution of inactivated normal rabbit 

 serum add 2 loopfuls of culture and inject 1 c.c. intraperitoneally. If 

 goats or horses are used in preparing the immune serum, this control 

 should be conducted with normal goat or horse serum. According to 

 Kolle, one loopful of virulent cholera culture is destroyed in the peri- 

 toneal cavity of a guinea-pig by 0.1 to 0.3 c.c. of normal rabbit's serum; 

 0.02 to 0.03 c.c. of normal goat's serum; 0.005 to 0.01 c.c. of normal 

 horse's serum. 



8. Control: A pig may be injected with 1 c.c. of a 1 : 100 dilution of 

 the immune serum, and with a loopful of some other microorganism, 

 such as Bacillus coli. This control, however, is not absolutely neces- 

 sary. 



An area of the abdominal wall of the guinea-pig about one inch in 

 diameter is shaved and cleansed with alcohol. After the injections have 

 been made the bacteriolytic phenomena are observed. 



In making this test fine capillary pipets are prepared and used for 

 withdrawing the peritoneal exudate. 



After permitting the animal to inhale a few drops of ether, to make 

 sure that it will not suffer, a small incision is made through the skin of 

 the abdomen. The capillary pipet, the large end of which is kept closed 

 with the index finger, is then quickly passed into the abdominal cavity. 

 The index-finger is released, and the tube is gently moved about and 

 withdrawn. As the result of capillary attraction sufficient exudate 

 usually passes into the tube without the aid of suction (Fig. 99). The 

 tube may be fitted with a rubber teat in case suction should be necessary. 

 Hanging-drop and smear preparations are made and studied microscop- 

 ically (Fig. 100). Stained smears are, however, less reliable and not so 

 useful in determining the degree of bacteriolysis (Fig. 102). 



It is best to withdraw the exudate immediately after injection, and 

 then at intervals of ten, twenty, thirty, forty, and sixty minutes. 



A hanging-drop preparation, made from the culture by emulsifying 

 a minute quantity in bouillon, should be on hand as a control in studying 



