PRACTICAL EXERCISES 71 



It will be found that more is now required. The cholesterin in the 

 serum neutralizes the action of some of the saponin. 



(4) (a) Put i c.c. of blood into each of two test-tubes. To one add 

 i c.c. of 2 per cent, aqueous solution of urea, and to the other 3 c.c. 

 Laking will take place in the second, whether this has been the case in 

 the first or not. 



(b) Repeat the experiment with a 2 per cent, solution of urea in 

 0-9 per cent. NaCl solution. Laking does not occur. This shows that 

 the urea in the first experiment did not act as a haemolytic agent. 

 Laking occurred because urea penetrates the corpuscles easily, and 

 therefore, although the freezing-point of the urea solution is not very 

 different from that of the NaCl solution, its actual osmotic pressure, 

 in relation to the envelopes of the corpuscles, is very much less, and the 

 laking is really water-laking. 



(5) Put some blood into a flask or test-tube, cork up, and let it stand 

 till it begins to putrefy. It becomes laked. The same occurs when 

 the blood is collected aseptically in a sterile tube and sealed up, although 

 it takes a longer time for the laking to become complete. 



(6) With blood containing nucleated corpuscles (necturus, frog or 

 chicken) diluted with isotonic salt solution, perform the following 

 experiments under the microscope : 



(a) With a glass fbd drawn to a fine point put a small drop of blood 

 on a slide, and near it a drop of distilled water. Carefully lower the 

 cover-slip and observe the interface with the microscope, first with the 

 low and then with the high power. Then mix and see complete laking. 

 Add a little methylene blue. Note that the nuclei still stain. 



(b) Place a small drop of a 3 per cent, solution of saponin in isotonic 

 salt solution on a slide, and near it a small drop of blood. Observe as 

 in (a). Repeat with a 2 per cent, solution of sodium taurocholate in 

 salt solution. If necturus corpuscles, which are splendid objects for 

 such experiments on account of their great size, have been used, 

 intracorpuscular crystallization of the haemoglobin may be observed. 



(c) Repeat (a) and (b) with mammalian blood. Note that the cor- 

 puscles swell before being laked by the saponin . If any of the corpuscles 

 are crenated, it may be seen that before being laked by the saponin 

 the crenations disappear, the corpuscles becoming round, while in the 

 taurocholate solution they may remain crenated till laking has occurred. 

 This indicates that the permeability of the envelopes is not affected in 

 the same way by the two laking agents. 



15. 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 microscopically. 



(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 put, leaving only 

 their ' ghosts.' A few of the corpuscles which come into contact witn 

 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. 



