72 THE CIRCULATING LIQUIDS OF THE BODY 



(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 0-5 c.c. dog's serum. 



(c) To C and C' add respectively o-i c.c. and 0-5 c.c. of 0-9 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. t 



(6) Heat i c.c. oi 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 tenj minutes, and repeat experiment (5), labelling the tubes D, D', 

 E, E', F, F'. Save the rest of the heated sera for (8). There is no 

 laking in any of the tubes, but probably agglutination in D, D', and 

 E, E'. (The complement is destroyed, but not the intermediary body 

 or amboceptor, or the agglutinin p. 28.) 



(7) Put half of the contents of tubes D, D', E, E', into four separate 

 test-tubes, and add to each 0-2 c.c. of rabbit's serum. If there is laking 

 now it is because the rabbit's serum contains complement. Save the 

 balance of D, D', E and E' for (8). 



(8) Allow 0'5 c.c. of ox serum to act at o C. on the rabbit's washed 

 corpuscles contained in 5 c.c. of the 5 per cent, suspension after removal 

 of the sodium chloride solution. The ox serum and rabbit's corpuscles 

 are separately cooled to o C. before being mixed, and the mixture is 

 then kept at o C. for one hour. Centrifugalize the serum off rapidly. 

 Label it ' Serum S.' To 0*2 c.c. of the original 5 per cent, suspension 

 of rabbit's washed corpuscles add o-i c.c. of this serum (labelling the 

 tube G), and put at 40 C. with a control-tube containing the same 

 amount of suspension plus salt solution instead of serum. Add the 

 rest of the serum S, cooled to o C., to the same cooled rabbit's cor- 

 puscles, and leave for a further period at o C. Then centrifugalize 

 rapidly, and to 0-2 c.c. of the original suspension of washed rabbit's 

 corpuscles add o-i c.c. of serum S (labelling the tube H), and put at 

 40 C. with a sodium chloride tube as control. There may be no 



* 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 corpuscles 

 obtainable from a rabbit. The injection should be made under the skin or, 

 better, into the peritoneal cavity of course, with aseptic precautions. It 

 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 centrif- 

 ugalize. 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. 



