Hwmolytic Activity of Chemical Substances. 287 



each of these variables must be controlled or measured : where a standard 

 blood suspension is used, (2) may be neglected. 



1. A solution of convenient strength in 0'95 per cent. NaCl of the hsemo- 

 lytic substance to be investigated is prepared : as a rule, a solution 4 c.c. of 

 which contains 50 mgmi. of the substance is employed. In the case of the 

 bile salts, a solution of about this strength is essential ; it is further very 

 convenient to have the solution so adjusted, as it facilitates calculation of 

 dilutions. From this solution dilutions are made as required. A series of 

 dilutions are made, e.g., 1-1000, 1-2000, 1-3000, 1-4000. 4 c.c. of each dilution 

 is placed in a series of tubes with thermometers, as described above. The 

 tubes are then placed in the water bath at, say, 40° C. ; with them is placed a 

 tube containing blood suspension. When the temperature of each tube has 

 reached the temperature of the bath, as indicated by the thermometers 

 carried by each, the blood suspension is added and the experiment commenced. 

 This requires care and practice to perform satisfactorily and is done as 

 follows: The tube containing the blood suspension is iixverted rapidly, to 

 mix the contents, and is then replaced in the bath. The pipette of 1 cc, 

 capacity with which the measured quantity of blood suspension is to be 

 added to each tube, is warmed by drawing up through it saline which has 

 been warmed to a moderate heat over a flame. It is then at once dipped 

 into the tube containing the blood suspension, of which 1 c.c. is drawn up ; 

 this is delivered without delay into the tube containing the strongest solution 

 of heemolytic agent (in the above series the 1-1000 tube). The time is noted ; 

 the process of adding blood suspension is repeated in the case of each of the 

 tubes containing dilutions of haemolytic agent, the moment of adding the 

 blood being observed in each case. A stop-watch is almost essential. The 

 operation requires to be carried out as quickly as possible. There should be 

 scarcely an appreciable loss of temperature in the contents of the tubes when 

 the blood is added. This result can be attained by speed and practice ; 

 although the method may appear clumsy it is open to fewer objections than 

 is the method of adding the suspension by upsetting a small tube in which it 

 is contained, and which is placed inside the large tube. It is possible to 

 carry out this operation for ten tubes within thirty seconds, and with a loss 

 of no more than 1/10° C. From the moment of adding the suspension the 

 temperature in each tube should remain constant. 



The moment of complete haemolysis in the case of any tube is decided 

 preferably by comparison with a fully hsemolysed control tube, which is 

 placed in the bath. The water bath being placed against an illuminated 

 screen, it is easy to compare the intensity of blackness with which a black 

 rod placed horizontally across the screen is seen, in the case of tube and 



