286 Mr, E. Ponder. A Method for Investigating the 



(6) Three water baths, for maintaining temperatures at about 3° C, 12° C. 

 and 30° — 50° C. respectively. These should each be capable of containing 

 about six of the tubes above mentioned in such a way that the degree of 

 haemolysis in the contents of the tubes may be seen without removing the 

 tubes from the bath. The baths are arranged so that the contents of the 

 tubes are seen against a screen lit by artificial light. 



Preparation of the Standard Blood Suspension, 



The suspension used in all the experiments is of an arbitrary, but con- 

 venient strength. 



It is prepared as follows : — 0'5 c.c. of blood is drawn from the fiuger ; the 

 blood must flow easily, and the finger must not be squeezed. This volume of 

 blood is added to a tube containing 15 c.c. of 1'5 per cent, citrated saline ; the 

 receiving -pipette should be rinsed with this saline, and drained, before 

 drawing up the blood. The contents of the tube are then centrifuged, the 

 clear supernatant fluid removed, and the tube again filled, this time with 

 95 per cent, sodium chloride. Again it is centrifuged. This process is 

 repeated, so as to make four washings in all. After the final washing, the 

 supernatant saline is removed, the tubes put in the centrifuge for a few 

 minutes, and the last drops of saline taken off the cells with a capillary 

 pipette. The cells are then added to 10 c.c. of 0'95 per cent, sodium chloride. 



The resulting blood suspension will keep for fully eighteen hours, but 

 should be used soon after preparation. In carrying out series of experiments, 

 it is important that the blood used should be derived from the same person, 

 e.g., from the experimenter. During more than a year's work, involving the 

 preparation of nearly a thousand such suspensions, and using the blood of the 

 same person, a variation of the suspension sufficiently great to be detected 

 has never occurred. It may, therefore, be taken that the suspension, if 

 carefully prepared, is reliably constant in strength ; it is, however, most 

 necessary that the cells be freed from all traces of serum. 



All the formulte given refer to this suspension : the modifications of the 

 formulae required if suspensions of other strength are used will be given 

 later. 



Technique. 



To investigate the hsemolytic activity of a haemolytic substance, the 

 following technique is employed. Since the activity of such 'a substance 

 depends on (1) the dilution of the substance, (2) the quantity of blood 

 suspension which it has to htemolyse, (3) the time which it takes to complete 

 this haemolysis, and (4) the temperature at which the experiment is conducted,. 



