896 EXPERIMENTAL INFECTION AND IMMUNITY 



3. Into a series of six test-tubes place 1 c.c. of the corpuscle emulsion and in- 

 creasing doses of toxin: 0.1 c.c., 0.2 c.c., 0.4 c.c., 0.8 c.c., 1 c.c., 2 c.c. Add 

 sufficient normal salt solution to bring the total volume to 3 c.c. 



4. Place 1 c.c. of the corpuscle suspension in 2 c.c. salt solution as a control to 

 make sure that the salt solution is isotonic. 



5. Heat a portion of toxin at 60 C. for an hour in a water-bath and set up a 

 similar set of tubes. 



6. Incubate all tubes for two hours at 37 C. 



7. Read results at the end of this time and again after the tubes have settled in 

 the refrigerator overnight. 



(a) Has hemolysis occurred in any of the tubes? 



(b) What is meant by hemolysis? 



(c) What constituent of tetanus toxin is responsible for hemolyzing 

 these cells? 



(d) Does this experiment show the selective affinity of a toxin for 

 certain cells? 



(e) How is tetanus toxin produced? 



(f) Does heat destroy the hemotoxic agent? 



EXERCISE 4. TOXINS (Continued) 

 EXPERIMENT 10. BOTULISM TOXIN 



1. Prepare toxin by cultivating the Bacillus botulinus in an alkaline bouillon 

 made in the form of an infusion from ham with the addition of 1 per cent, of glucose, 

 1 per cent, of peptone, and 1 per cent, of sodium chlorid. Strict anaerobic cultures 

 should be grown for four weeks and filtered through a Berkefeld filter. The toxin 

 may be preserved in brown, sealed vials, and kept on ice, or in a dried form in vacuum. 



2. Dilute 0.2 c.c. of the toxin with 3 or 4 c.c. salt solution and administer, per os, 

 to a cat by means of a small catheter passed into the stomach. 



3. Inject 0.1 c.c. of the toxin subcutaneously in the abdominal wall of a rabbit. 



4. Observe animals closely for several hours following administration of the toxin, 

 for some products are so highly toxic that symptoms may appear within a few hours. 



(a) What are the symptoms of botulism infection? 



(b) Do these symptoms show any selective action of botulism toxin 

 for certain tissues? 



(c) Autopsy the animals. Are there any gross tissue changes? 



EXPERIMENT 11. DYSENTERY TOXIN 



1. Inoculate a flask of moderately alkaline bouillon with a culture of dysentery 

 bacilli, preferably of the Shiga-Kruse type. Inoculate at 37 C. for two weeks and 

 pass it through a bacterial filter. 



2. Inoculate three rabbits intravenously with 1, 2, and 5 c.c. of the filtered toxin. 



3. Give a fourth rabbit 2 c.c. of the toxin by mouth through a small catheter 

 passed into the stomach. 



4. Observe the animals very closely. 



