154 LABORATORY COURSE IN SERUM STUDY 



After one half hour at 37 C. add 0.5 c.c. of 5 per cent rabbit cells 

 to all tubes, incubate one hour, and observe haemolysis. * 



Tube 8 is inserted, of course, to make certain that the toxin does 

 not lose its haemolytic power during the half hour of preliminary incuba- 

 tion, and Tube 9 to show that horse serum is not of itself haemolytic. 



TETANOSPASMIN 



Students should work in groups of three or four in performing 

 the following experiments. Fresh tetanus toxin is obtained from 

 a department of health or one of the commercial laboratories with 

 the lethal dose for guinea pigs of 350 grams worked out. Tetanus 

 antitoxin can be procured from the same source or in the market. 



4. Inject a guinea pig of approximately 350 grams into the thigh of 

 one hind leg with five lethal doses of the tetanus toxin. 



B. A similar guinea pig at the same time is injected with twice the 

 dose, i.e. ten lethal doses of tetanus toxin, which has been mixed with 

 one antitoxin unit, 1 the mixture having been allowed to stand at room 

 temperature for twenty minutes. 



C. Grind up five lethal doses of the tetanus toxin with half of one 

 cortex of fresh guinea pig brain, adding salt solution to the amount of 

 3 or 4 c.c. during the process. This mixture is allowed to stand at 

 room temperature for two or three hours. It is then centrifugalized 

 and the supernatant fluid injected into a third pig. 



The pigs are kept under close observation for 6 days. 



Materials required for each student for the tetanolysin ex- 

 periments: Tetanus toxin about 4 c.c. 

 Tetanus antitoxin about 0.1 c.c. 

 Normal horse serum about 0.1 c.c. 

 5 per cent rabbit-cell suspension 8.0 c.c. 



Materials for each group of 3 or 4 students for the tetanospas- 

 min experiment : 



3 guinea pigs of about 350 gms. each. 



20 lethal doses of tetanus toxin of known toxicity. 



Tetanus antitoxin, one unit. 



\ fresh guinea pig brain. 



1 The antitoxin unit is established by the United States Hygienic Laboratory 

 (Bulletin 43) on principles similar to those used for diphtheria antitoxin and 

 represents ten times the amount of antitoxin necessary to completely neutralize 

 100 minimal lethal doses of a standard toxin injected into a guinea pig of 350 

 grams. 



