PRACTICAL EXERCISES 63 



12. Haemolysis and Agglutination by Foreign Serum. (i) To a 

 small quantity of rabbit's blood add an equal volume of dog's serum. 

 Mix and let stand at 40 C. The colour of the blood is soon darker 

 than before, and it can be seen to be laked. Examine microscopi- 

 cally. 



(2) Place a small drop of rabbit's blood and a somewhat larger 

 drop of the dog's serum on a slide, near, but not quite in contact 

 with, each other. Now put on a cover-slip, so that the drops just 

 come together, and examine at once with the microscope with a 

 moderately high power. Where the two drops mingle, the red 

 corpuscles will be seen first to become agglutinated into groups, and 

 then to fade out, leaving only their ' ghosts.' A few of the corpuscles 

 which come into contact with the, as yet, undiluted serum may be 

 entirely dissolved. 



(3) Heat some of the dog's serum to 60 C. for ten minutes, and 

 repeat (i) and (2). No laking will now be produced in the rabbit's 

 corpuscles, but agglutination may be observed as before. 



(4) Repeat (i) and (2) with dog's blood and rabbit's serum. The 

 blood will not be laked, although sometimes the dog's corpuscles 

 may become crenated. There will be no agglutination. 



(5) With a 5 per cent, suspension of rabbit's washed corpuscles 

 perform the following experiments :* 



Put into each of six small test-tubes i c.c. of the suspension. 

 Label the tubes A, A', B, B', C, C'. 



(a) To A and A' add respectively o'i c.c. and 0^5 c.c. ox serum. 



(b) To B and B' add respectively o'i c.c. and o'5 c.c. dog's serum. 



(c) To C and C' add respectively o* i c.c. and 0*5 c.c. of o'g per cent, 

 sodium chloride solution. 



Put all the tubes in a bath at 40 C. Compare the amount of 

 laking and agglutination in the various tubes at intervals of two 

 minutes or less. Repeat (a), (b), and (c} with guinea-pig's washed 

 corpuscles and serum of ox and dog. Determine which of these 

 sera has the strongest haemolytic power.f 



(6) Heat i c.c. of ox and dog's serum respectively to 56 C., keeping 

 it at that temperature, or not more than a couple of degrees above 

 it, for ten J minutes, and repeat experiment (5), labelling the tubes 



* The material obtained from one medium-sized dog, two rabbits, and 

 one guinea-pig is enough for fifty or sixty students, working together in 

 sets of two, to perform experiments (5) to (8). In order to obtain a serum 

 more strongly haemolytic for rabbit's corpuscles than normal dog's serum, 

 a dog may be ' immunized ' by previous injection of all the washed cor- 

 puscles obtainable from a rabbit. The injection should be made under 

 the skin or, better, into the peritoneal cavity of course, with aseptic 

 precautions. Tt should be repeated not less than twice, with an interval 

 of ten days between the successive injections, and the dog's blood should 

 be drawn off about ten days after the last injection. 



f To determine the amount of laking at any given moment, drop the 

 small test-tubes into the metallic centrifuge cups after shaking them up, 

 and centrifugalize. A very short time is sufficient to separate a clear 

 supernatant liquid, from the tint of which the extent of the haemolysis 

 can be deduced. Before replacing the tubes in the thermostat, they should, 

 of course, be shaken up. Small test-tubes of about 8 mm. internal diameter 

 and short enough to go conveniently into the centrifuge cups are the most 

 serviceable. 



J For exact work a longer time is recommended. But for the student 

 the time is made as short as possible, and it is only in exceptional cases 

 that ten minutes is not enough. 



