BACTERIAL VACCINES 837 



is added, the second with 0.2 c.c. serum and dose of corpuscles. Shake tubes and 

 incubate for two hours. Make a preliminary reading and again after tubes have 

 settled twenty-four hours in the refrigerator. 



3. The first control should be completely hemolyzed, indicating that a sufficient 

 lytic dose of toxin was employed. 



(a) What constituent of tetanus toxin has a marked affinity for 

 erythrocytes? 



(b) Is this agent thermostabile? How can you determine this? 



(c) What role does it play in tetanus intoxication? 



(d) How is this hemotoxic agent neutralized by tetanus antitoxin? 



(e) Would anti-tetanospasmin neutralize the hemotoxic activity 

 of tetanus toxin? 



(f) Would diphtheria antitoxin neutralize this hemotoxic agent? 

 If not, why not? 



(g) Explain the mechanism of neutralization of a toxin by antitoxin 

 in vitro? Is it the same as that occurring in vivo? 



EXPERIMENT 48. ANTISTAPHYLOLYSIN 



1. Prepare a staphylolysin by growing a culture of Staphylococcus aureus in 

 bouillon for two or three weeks. Pass through a Berkefeld filter and preserve the 

 filtrate with 0.5 phenol. Determine the lytic dose for 1 c.c. of a 1 per cent, sus- 

 pension of rabbit corpuscles. 



2. .Secure serum from a rabbit immunized with staphylococci. Heat at 56 C. 

 for thirty minutes. 



3. Secure normal horse serum. Heat it at 56 C. for thirty minutes. 



4. Secure normal rabbit serum. Heat at 56 C. for thirty minutes. 



5. Into a series of six small test-tubes place the lytic dose of staphylolysin and 

 increasing amounts of rabbit immune serum as follows: 0.001, 0.005, 0.01, 0.05, 0.1, 

 0.2 c.c. Arrange a similar series, using normal horse serum and normal rabbit serum. 

 Prepare a control containing the lytic dose of toxin. Add 1 c.c. of a 1 per cent, sus- 

 pension of rabbit cells to each tube and sufficient normal salt solution to make the 

 total volume equal 2 c.c. Shake each tube gently and incubate at 37 C. for two 

 hours. 



6. Inspect the tubes. The smallest amount of normal horse serum inhibiting 

 hemolysis is taken as 1, or the unit. Compare the values of the immune and normal 

 rabbit serums with this unit. 



(a) Why is normal horse serum adopted as the standard? 



(b) What is antistaphylolysin? 



(c) What are the various agents produced by staphylococci and re- 

 sponsible for the lesions and symptoms of Staphylococcus infections? 



(d) Would the antilysin neutralize the leukocidin? 



(e) Explain the mechanism of lysin-antilysin action. 



(f) Which role does the lysin play in Staphylococcus infections? 



(g) Of what value would be the titration of antistaphylolysin in the 

 serum of the patient? 



