PHYSIOLOGICAL CHEMISTRY 463 



first of all necessary to take blood corpuscles that have been washed free of 

 adherent serum. 1 



5. Determine the minimum amount of a 0*3 per cent, saponin solution necessary 

 to produce laking of icashed dog's corpuscles in five minutes at room temperature. 



Label four test tubes A, B, C, D, and place in each 2 c.c. of 10 per cent, dilution 

 of dog's washed blood corpuscles in 0'9 per cent. NaCl solution. Of a 0'3 per cent, 

 solution of saponin in 0'9 per cent. NaCl solution, add to A 0*05 c.c. ; to B 

 O'l c.c. ; to C 0-15 c.c. ; to D 0'2 c.c. Immediately fill each test tube with 

 0'9 NaCl solution and without shaking allow to stand for five minutes. Then 

 note the tube which just shows complete laking. Repeat the observation 

 taking amounts of saponin in l/100th of a c.c. between the amounts in the 

 previous trial, which did and which did not show laking (i.e. suppose trial 

 gave laking with 0*15 c.c. but not with O'lO, then in this experiment use 

 0.11 c.c. saponin in A ; 0'12 c.c. in B t and so on). 



6. Demonstrate the antidotal effect of blood serum on saponin laking. Place 

 2 c.c. of a 10 per cent, suspension of dog's washed blood corpuscles in four 

 marked test tubes: add to each 2 c.c. of dog's blood serum and mix. Then 

 add minimal laking amount of saponin solution (determined as in Experiment 5) 

 to A and to the others amounts increasing by 0'05 c.c. Fill up the test tubes 

 with 0-9 per cent, sodium chloride solution and, after standing for five minutes, 

 see in which tube laking has occurred. The antidotal effect of the blood serum 

 will be clearly shown. 



There are some very peculiar differences between the action of laking agents. 

 Thus saponin evidently acts on some constituent of the envelope which is closely 

 related to cholesterol (or lecithin), for if a saponin solution be shaken with 

 cholesterol its haemolysing effect will become greatly reduced. In its action on 

 the corpuscular envelope the saponin either dissolves the cholesterol or forms a 

 compound with it which is soluble in the surrounding fluid, and in this way 

 makes the envelope so permeable that the haemoglobin escapes. It is of interest, 

 in this connection, to note that if the mixture of blood and saponin be examined 

 under the microscope, the corpuscle will be seen to swell somewhat before 

 haemolysis occurs, showing that its permeability towards water is first of all 

 lowered. The electrical conductivity increases somewhat during laking by a 

 minimal dose of saponin, probably due to the escape of haemoglobin, but if 

 at this stage more saponin be added, the electrical conductivity still further 

 increases, showing probably that some compound of strorna and inorganic salts 

 has been broken down (Stewart). With bile salts, on the other hand, there 

 is no preliminary swelling of the corpuscles ; their haemoglobin contents are set 

 free immediately. Ether does not merely act by dissolving out lipoid, for ether 

 saturated with cholesterol or with the lipoid substances of corpuscles themselves 

 still causes laking. 



Bio-chemical Laking, 



Perhaps the most interesting haemolytic bodies are those which become 



x To wash blood corpuscles free of adherent serum a good centrifuge is 

 necessary. The defibrinated blood is first of all centrifuged and the serum 

 removed with a pipette. The centrifuge tube is then filled up with 0'9 per 

 cent, sodium chloride solution and, after shaking, placed in the centrifuge. 

 This process is repeated at least three times and, for more particular work, 

 even more. 



