l86 HAEMOLYSIS METHODS OF RESEARCH 



is to administer chloroform, and insert a cannula into the carotid 

 artery ; an easier method is to stick a small piece of capillary 

 glass tubing through the wall of the heart whilst it is still beating, 

 the animal being, of course, under chloroform. In either case the 

 blood is led into a sterile tube, allowed to clot preferably in the 

 incubator, as the retraction of the clot is thereby increased and 

 the clear serum 'withdrawn by means of a sterile pipette, and 

 stored in sterile tubes or small flasks, which may be plugged with 

 cotton- wool or hermetically sealed. Where the serum is to be heated 

 to destroy complement this may be accomplished in a thermostat, 

 or more simply by sealing it in a narrow tube which is tied to the 

 bulb of a thermometer, and placed in a beaker of warm water, and 

 the temperature regulated by hand by the application and removal 

 of a spirit-lamp. By this means any desired temperature can be 

 maintained within very close limits, and the necessity for main- 

 taining several thermostats or of altering the regulator is 

 removed. 



The emulsion of corpuscles to be employed in the actual testing 

 is prepared as above, and must be rewashed at least four times, 

 since it is found that traces of complement may remain after three 

 washings. The emulsion is usually made up so that it contains 

 5 per cent, of corpuscles. This is readily accomplished by per- 

 forming the last centrifugalization in a graduated tube, going on 

 until the volume is constant. The bulk of the corpuscles is then 

 read off, and, the necessary amount of normal saline solution 

 being added, the corpuscles thoroughly mixed in. It is advisable 

 that aseptic precautions should be observed throughout, but as 

 this is troublesome it can often be omitted. It must be remem- 

 bered, however, that many common bacteria produce haemolysis, 

 and that if the mixtures of corpuscles, serum, etc., be incubated 

 for long periods fallacies may arise from this cause. 



The actual experiment in its simplest form is carried out as 

 follows : The necessary amounts of 5 per cent, emulsion of 

 corpuscles, heated serum, and complement are placed in a narrow 

 test-tube, and in most cases normal saline solution is added to 

 bring the whole up to a definite volume. This is now incubated 

 for two hours, being stirred or shaken once or twice in the mean- 

 time. It is now removed and placed in a vertical position in the 

 ice-chest for twelve hours or so and examined. If there is com- 

 plete haemolysis the fluid will be deeply coloured, and there will be 

 no sediment, or only a minute deposit of stromata. With partial 

 haemolysis the fluid will be less deeply-coloured, and there will be 



