308 GENERAL MICROBIOLOGY 



EXERCISE 6. THE PREPARATION OF TETANUS TOXIN 



Apparatus. Dextrose broth; two 100 c.c. Erlenmeyer 

 flasks; paraffin oil; 5% phenol; Berkefeld filter. 



Culture. B. tetani. 



Method. 1. Place 50 c.c. of dextrose bouillon in each 

 of two 100 c.c. Erlenmeyer flasks, plug and boil gently 

 two or three minutes over a flame. 



2. Cover the bouillon with a layer of paraffin oil about 

 5 mm. deep and heat in the autoclav. 



3. After cooling, inoculate the bouillon with B. tetani 

 and incubate about two weeks. 



4. Examine the culture microscopically to determine 

 the absence of contamination, add sufficient 5% phenol 

 to make a 0.5% solution and filter through a Berkefeld filter. 



5. Incubate about 1 c.c. of the filtrate in 10 c.c. of 

 dextrose broth under anaerobic condition, for forty-eight 

 hours to make sure that the filtrate is sterile. 



6. The filtrate, if sterile, is to be used in immunizing a 

 rabbit for the production of antitoxin. 



Note. A fairly potent toxin will kill a guinea pig in a 0.001 c.c. 

 dose. The toxin in solution is very unstable and should be kept in a 

 tightly stoppered bottle in a cool dark place. It may be kept for 

 several months by precipitating with a saturated solution of ammo- 

 nium sulphate and drying in vacuo over sulphuric acid. 



REFERENCES 



MARSHALL: Microbiology (1911), pp. 480-484. 



BESSON: Practical Bacteriology, Microbiology and Serum Therapy 



(1913), pp. 536-548. 

 KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 115, 



234, 720, 819. 

 ZINSSER: Infection and Resistance (1914), pp. 41, 107, 131-133. 



