330 COLLECTED STUDIES IN IMMUNITY. 



II. The Method of Making Haemolytic Experiments. 

 General Considerations. 



With a little practice the quantitative estimation of haemolysis 

 proves very simple. The two fundamental points, entire haemolysis 

 (complete), and no haemolysis whatever (0), are usually very readily 

 recognized. By " trace" we mean the occurrence of a faint zone 

 of solution observed just above the cells by gently agitating the 

 test-tube. The estimation of complete haemolysis only then offers 

 difficulties if considerable agglutination has occurred, so that the 

 fluid when shaken is clouded by the clumped stromata. Such cases in 

 themselves are poorly adapted for quantitative studies because at 

 times the rapid agglutination may purely mechanically prevent the 

 escape of the haemoglobin and so simulate an absence of haemolysis. 



In this respect according to our experiences the greatest diffi- 

 culties are presented by dog blood-cells and the specific immune 

 sera (derived from goats) against these. This is still more the case in 

 such sera derived from rabbits. It often happens, before even a trace 

 of haemolysis has occurred, that the dog blood-cells are agglutinated 

 and fall to the bottom of the test-tube. Goose blood and specific 

 immune serum behave similarly. In these cases it is necessary by 

 means of frequent shaking to separate the agglutinated blood-cells so 

 that the haemoglobin is given chance to escape. 



In those cases in which the usual method of describing the degree 

 of solution does not suffice, and accurate quantitative determinations 

 of the amount of blood-cells dissolved are desired, one makes use of 

 a colorimetric procedure devised by Madsen in which a color comparison 

 is always made by dissolving blood-cells in water. 1 



Agglutination is usually easily recognized on shaking up the sedi- 

 mented blood-cells. It becomes very evident when the specimens 

 of blood are shaken and one then compares the rapidity with which 

 the blood-cells settle to the bottom. This is always greater with 

 agglutinated blood-cells. 



In general a 5% suspension of the blood-cells in 0.85% salt solu- 

 tion has proven best adapted for haemolytic experiments. 1 to 2 cc. 

 of such a mixture in each test-tube is sufficient for most tests. When 

 material is scanty one can use amounts very much smaller, though 

 usually this will be at the expense of accuracy. In this case, of 



1 See Madsen, Zeitschrift fur Hygiene, Vol. 32, 1899. 



