848 EXPERIMENTAL INFECTION AND IMMUNITY 



EXPERIMENT 70. SERUM HEMOLYSIS IN VITRO 



1. Secure 2 c.c. of blood from the ear of a rabbit which has received at least two 

 intravenous injections of sheep cells. Separate the serum and divide into two 

 portions. Inactivate one portion (A) by heating in a water-bath for half an hour at 

 56 C. 



2. Place 0.1 c.c. and 0.2 c.c. of the fresh unheated serum of portion (B) in two 

 test-tubes. Likewise the same amounts of the heated serum (A) hi two more tubes. 

 Add 1 c.c. of a 1 per cent, suspension of washed sheep cells to each tube and sufficient 

 salt solution to bring the total volume to 4 c.c. As a control, place 1 c.c. of corpuscle 

 suspension and 3 c.c. salt solution in a tube. Shake gently, and incubate for an 

 hour. 



(a) Which tubes show hemolysis? 



(b) What substances are concerned in serum hemolysis? 



(c) Why did hemolysis occur with the unheated and not with the 

 heated serum? What is the meaning of inactivatwn of a serum? 



(d) Why is this called serum hemolysis? 



(e) What is the appearance of the control tube? Why is this con- 

 trol included? What would have happened if a hypotonic salt solution 

 had been used? 



EXPERIMENT 71. SERUM HEMOLYSIS IN Vivo 



1. Immunize a rabbit with three intravenous injections of 5 c.c. each of a 10 per 

 cent, suspension of washed cat erythrocytes in sterile salt solution. Give the in- 

 jections each day; four days after the last injection the rabbit blood is titrated and 

 usually contains a fair amount of anticat hemolysin. 



2. Heat 2 c.c. of the immune serum at 56 C. for thirty minutes and inject into 

 the external jugular vein of a cat. 



3. Place the animal in a metabolic cage and collect the urine. 



4. After two or three days, autopsy, removing the spleen, liver, and kidneys. 

 Place hi 5 per cent, formalin and prepare and stain sections with hematoxylin and 

 eosin and Giemsa solution. 



(a) Has blood destruction occurred? What are the evidences? 



(b) Would hemolysis occur in the test-tube with a heated immune 

 serum? If not, why not? 



(c) How then do you explain hemolysis in vivo with a heated serum? 



(d) Examine sections of the spleen, liver, and kidneys. Are there 

 any evidences of phagocytosis of blood-cells, focal necrosis, and nephritis? 

 Explain the probable mechanism of the production of these changes. 



EXERCISE 28. AMBOCEPTORS AND COMPLEMENTS. HEMOLYSINS 

 EXPERIMENT 72. TITRATION OF A HEMOLYTIC AMBQCEPTOR 



1. Secure 1 or 2 c.c. of blood from the ear of a rabbit which has been immunized 

 with injections of sheep cells. Separate the serum and inactivate by heating to 56 



