CONCENTRATION OF ANTIBODIES 129 



the blood into the other body fluids in animals passively immunized by the with- 

 drawal of large quantities of blood, and the injection of a corresponding amount of 

 warm, defibrinated blood from an actively immunized animal. 



The body fluids were secured under as nearly aseptic conditions as possible. The 

 animal was anesthetized with ether, and kept in a state of complete anesthesia, by the 

 administration of the vapor through a trachea cannula or through a tube introduced 

 through the larynx. The neck lymphatics were then isolated, and small, sterile, glass 

 cannulae provided with sterile, rubber tubing were inserted. If there was no free flow 

 of lymph, the neck was gently massaged. The lymph was never allowed to come in 

 contact with the air of the room, for as soon as it filled the cannula and a part of the 

 rubber tubing, it was drawn off by means of a fine, sterile Pasteur pipette, and placed 

 in a dry, sterile test tube plugged as for bacteriological work. The lymph was allowed 

 to coagulate spontaneously in the test tube, and was then defibrinated, and the delicate 

 coagulum removed. 



The thoracic duct was tied off at the same time as the isolation of the neck lym- 

 phatics so that the lymph formed during the experiment was retained in the duct. Usually 

 the lymph from this duct was not collected until the animal had been bled to death, 

 altho sometimes it was collected simultaneously with the neck lymph. The routine 

 method was to draw the lymph by means of a Pasteur pipette provided with a bulb, so 

 that the fluid never came in contact with the air at all. This fluid was also defibrinated. 



The pericardial fluid was never collected until after the death of the animal by very 

 complete bleeding from the arteries and veins of the neck. The thorax was opened 

 by removing the sternum, a small hole was cut into the pericardium, and the fluid was 

 removed by means of a sterile Pasteur pipette. 



We found it a good plan in our experiments to suspend the animal by the jaws for 

 a few minutes before attempting to withdraw the cerebrospinal fluid. This drained 

 away the blood from the head and made admixture of this fluid with blood less likely. 

 Our method was to open the dura between the first and the second cervical vertebrae, 

 and then remove the fluid by means of the Pasteur pipette. The end of the pipette 

 must be well rounded in the flame, otherwise rupture of the delicate blood vessels of 

 the meninges is likely to follow. 



The method of collecting the aqueous humor was simple and easy. It consisted 

 in thrusting a sharp pointed Pasteur pipette into the anterior chamber of the eye 

 through the cornea, and allowing the aqueous humor to flow into it, largely by the 

 tension within the eyeball. A little suction sufficed to remove the last drop of the fluid. 



The serum was secured from blood drawn when the animal was bled to death, 

 and in most cases was freed from the corpuscles at once. 



Careful notes were made in regard to the condition of the fluids, and in most 

 cases where there was any admixture of blood, the fluid was discarded. 



HEMOLYSIS AND HEMAGGLUTININS. 



Methods. The study of the hemolysins and hemagglutinins in normal* animals 

 was made on dogs only. The animals utilized were brought in from various parts of 

 the city. Most of the tests were made with rabbit corpuscles in 5 per cent suspension 

 in 0.9 per cent NaCl solution. In some cases rat and horse corpuscles were used. 



*The term "normal" means animals which had not previously been immunized by us. We had 

 no way of knowing what their history had been previous to coming to the laboratory. 



