102 LABORATORY GUIDE IN BACTERIOLOGY 



a) Make a solution of i g. Witte's peptone in 100 c.c. 

 of water. 



b) Distribute in three small Erlenmeyer flasks, and 

 sterilize in autoclav. 



c) Inoculate one of these flasks with Sp. chol. asiat. 

 and B. chol. suis or any other motile bacillus. 



d) Incubate at 37 for 18-24 hours. Make a stained 

 preparation from the surface of the liquid. 



e) After that time, take one loopful from the surface, 

 inoculate the second flask, and incubate as before. 



/) After 18-24 hours, make a stained preparation 

 from the surface of the second flask, and examine for 

 spirilla. 



g) Transfer a loopful from the surface of the second 

 flask to the third one, and incubate as before. 



h) After 18-24 hours, again stain and examine for 

 spirilla. By this time usually a film has formed which 

 contains the spirilla in practically pure culture. How is 

 this phenomenon explained, and what is its epidemiologi- 

 cal value ? 



5. Special study D. Inoculate a pigeon intramuscu- 

 larly with 0.5 c.c. of a broth-culture of Sp. Metchnikom. 

 The breast of the pigeon is laid bare, washed with mercuric 

 chlorid and alcohol, and the syringe is plunged into the 

 muscle-fibers and discharged. After death, note the 

 peculiar appearance, resembling that of boiled beef. 

 Make stained preparations from blood and muscle-juice, 

 and examine for spirilla. 



