924 EXPERIMENTAL INFECTION AND IMMUNITY 



EXPERIMENT 70. -THE HEMOLYTIC INFLUENCE OF ACIDS AND ALKALIS 

 The object of this experiment is to demonstrate the hemolytic 

 activity of acids and alkalies as bearing upon the question of clean glass- 

 ware in hemolytic work, and particularly complement-fixation tests. 



1. Into a series of six test-tubes place increasing amounts of a solution of hydro- 

 chloric acid prepared by diluting 1 c.c. of the normal solution with 99 c.c. of normal 

 salt solution: 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 c.c. respectively. 



2. Into a second series of six test-tubes place increasing amounts of a solution of 

 sodium hydroxid prepared by diluting 1 c.c. of the normal solution with 99 c.c. of 

 normal salt solution: 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 c.c. respectively. 



3. Make the total volume in each tube equal 2 c.c. with the addition of normal 

 salt solution. 



4. To each tube and a normal salt solution control add 1 c.c. of a 2| per cent, 

 suspension of washed sheep cells. Mix and stand aside for an hour or two. 



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



(b) What are the smallest amounts of acid and alkali producing 

 complete hemolysis in this experiment? 



(c) Of what practical significance are these results? 



(d) How should test-tubes be prepared for hemolytic work? 



EXPERIMENT 71. 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) in 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 3 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 inactivation 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 72. 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- 



