854 EXPERIMENTAL INFECTION AND IMMUNITY 



EXPERIMENT 82. GENERAL PROPERTIES OF COMPLEMENT 



1. Secure antisheep amboceptor the hemolytic unit of which is known by pre- 

 vious titration. 



2. Prepare a 2^ per cent, suspension of washed sheep corpuscles. 



3. Into a series of four test-tubes place the following: 

 Tube 1 : 1 c.c. of fresh guinea-pig serum diluted 1 : 20. 



Tube 2: 1 c.c. of guinea-pig serum (1:20) which has been kept at room tempera- 

 ture for two days. 



Tube 3: 1 c.c. of guinea-pig serum (1:20) three days old. 



Tube 4: 1 c.c. of guinea-pig serum (1:20) five days old. 



Add 1 c.c. of a 23^ per cent, suspension of sheep corpuscles, two hemolytic units 

 of antisheep amboceptor, and sufficient normal salt solution to each tube. Shake 

 gently and incubate at 37 C. for one hour. 



4. Into a series of six test-tubes place 1 c.c. of fresh guinea-pig complement serum 

 diluted 1:20. Place these in a water-bath at 56 C. At intervals of ten, twenty, 

 thirty, forty, fifty, and sixty minutes remove a tube, add 1 c.c. corpuscle suspension, 

 two units of amboceptor, and sufficient salt solution. Shake gently each tube and 

 incubate for one hour at 37 C. 



(a) Record the results. Does hemolytic complement deteriorate 

 readily at room temperature? 



(b) What are complementoids? 



(c) What practical significance has this experiment? Should a 

 complement serum be fresh when used in hemolytic work? 



(d) Is complement thermolabile? How long does it take to destroy 

 a diluted complement at 56 C.? 



(e) In inactivating a serum why do we not use a higher temperature? 



EXERCISE 33. AMBOCEPTORS AND COMPLEMENTS (Continued) 

 EXPERIMENT 83. TITRATION OF HEMOLYTIC COMPLEMENT 



1. Prepare 20 c.c. of complement 1:20; prepare a 2% per cent, suspension of 

 sheep cells. 



2. To a series of 10 test-tubes add increasing doses of diluted complement serum: 

 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, and 1.5 c.c.; add \Y 2 units of hemolytic ambo- 

 ceptor (determined by previous titration) ; 1 c.c. of corpuscle suspension and sufficient 

 salt solution to bring the total volume in each tube to 3 c.c. 



3. Shake gently and incubate for one hour at 37 C. 



(a) Record the results. What is the complement unit of this serum? 



(b) What animal serum is best adapted for complement in hemolytic 

 work? 



(c) Is the complement content of the serums of different animals 

 constant? Does it vary in animals of the same species? In the same 

 animal at different times? 



