TOXINS 819 



(c) Why does the use of diphtheria antitoxin make the test con- 

 clusive? Would tetanus antitoxin be capable of neutralizing diphtheria 

 toxin? 



EXPERIMENT 8. TETANUS TOXIN 



Tetanus toxin is composed of two distinct poisons of different prop- 

 erties. One, tetanospasmin, has a great affinity for the central nervous 

 system, and is largely responsible for the symptoms of tetanus infec- 

 tion (neurotoxic) ; the second, tetanolysin, is thermolabile and is hema- 

 toxic. Tetanus toxin is very labile, and when in solution soon becomes 

 attenuated. For these experiments it is necessary to use either fresh 

 toxin or that which has been recently precipitated and dried. 



1. Secure some dried tetanus toxin and dissolve in sterile salt solution. The 

 toxin may be secured from an antitoxin laboratory and preserved indefinitely in the 

 refrigerator. Since the strength varies with different products, the lethal dose for a 

 300-gram guinea-pig should be ascertained from the laboratory furnishing the toxin. 



2. Secure 2 grams of fresh normal guinea-pig brain and liver. Crush in separate 

 sterile mortars. 



3. Add a lethal dose of fresh tetanus toxin and 5 c.c. sterile salt solution to each. 

 Mix thoroughly and place in the incubator for an hour. Remove and carefully trans- 

 fer to sterile centrifuge tubes. Centrifuge thoroughly. 



4. Inject two 300-gram guinea-pigs subcutaneously in the median abdominal 

 line with the supernatant fluids. 



5. Inject a third guinea-pig with a similar dose of toxin and 5 c.c. salt solution 

 (control). 



6. Mark pigs carefully and observe for several days. 



(a) What are the symptoms of tetanus? 



(b) To what constituents of tetanus toxin are the chief symptoms due? 



(c) Does the pig which received the toxin-brain mixture show symp- 

 toms? How do you explain the result? 



(d) Does the pig which received the toxin-liver mixture show symp- 

 toms? How do you account for the result? 



(e) Is the tetanus bacillus characterized by its toxicity or aggressive- 

 ness? 



(f) Is there a period of incubation before symptoms develop and 

 why? 



(g) Autopsy the animal. Are there any definite lesions of the tissues 

 and internal organs? 



EXPERIMENT 9. TETANUS TOXIN 



1. Secure some fresh tetanus toxin. 



2. Prepare a 1 per cent, emulsion of washed sheep corpuscles (washed three times 

 in an excess of normal salt solution). 



