62 A MANUAL OF PHYSIOLOGY 



of bile salts (or of sodium taurocholate) in 0*9 per cent. NaCl solution. 

 Laking occurs in all. 



(b) To 5 c.c. of blood add 0*5 c.c. of a 3 per cent, solution of saponin 

 in 0*9 per cent. NaCl solution, and put the mixture at 40 C. Laking 

 soon occurs. 



(c) Using a 10 per cent, dilution of blood (blood to which nine 

 volumes of NaCl solution have been added) or a 5 per cent, suspension 

 of washed corpuscles in NaCl solution (i.e., a suspension of corpuscles 

 which have been washed free from serum by being repeatedly mixed 

 with NaCl solution and centrifugalized), determine the minimum 

 dose of o'3 per cent, saponin solution which will just cause complete 

 laking. Keep the tubes at about 40 C., and observe them from 

 time to time. Now add to some of the 10 per cent, dilution or the 

 5 per cent, suspension of blood an equal volume of serum from the 

 same kind of blood, and repeat the determination of the minimum 

 dose of saponin necessary for laking. 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 

 o'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-rod 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 

 corpuscles 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. 



