438 WILLIAM H. F. ADDISON 



EXPERIMENTAL PROCEDURE 



The pigeon blood was prepared for injection in the following 

 manner. The pigeon was bled from the jugular veins into 2 

 per cent sodium citrate in 0.85 per cent sodium chloride solution. 

 Usually 10 to 12 cc. of blood was available, and about 25 cc. of 

 the citrate solution was used to receive the blood. This was 

 centrifuged for fifteen minutes, and the supernatant fluid pipetted 

 off. The corpuscles were next shaken up with 2 cc. of the citrate 

 solution, and then the centrifuge tube filled with 0.95 per cent 

 NaCl solution. The corpuscles were washed twice again, each time 

 first shaking up the corpuscles with a small amount of the citrate 

 solution, and the tube then filled with the 0.95 per cent NaCl 

 solution. Finally, the volume of the washed corpuscles was 

 made up to 10 to 12 cc. with the 0.95 per cent NaCl. Under 

 this treatment there was no visible hemolysis and under the 

 microscope there was seen no clumping of the red blood-cells. 

 Usually 10 cc. of this was injected into a superficial ear vein of 

 the rabbit. The rabbits were taken at varying intervals after 

 injection, from one hour to forty-eight hours after injection. 

 After etherizing the rabbit, the animal was bled through the 

 inferior vena cava and normal saline transfused through the 

 thoracic aorta. After the spleen was partly washed out, as 

 shown by its paler color, the fixing fluid was run in, and attempts 

 were made to distend the spleen as much as possible. The fixing 

 fluid used was a mixture of commercial formalin, made neutral 

 by previously adding magnesium carbonate in sufficient quantity 

 to form a thin layer at the bottom of the stock bottle, and 3 per 

 cent aqueous solution of potassium bichromate. The two fluids 

 were mixed, just before using, in the proportion of one part of 

 neutral formalin to nine parts of the bichromate. Small pieces 

 of the organ were subsequently fixed for a period of twenty-four 

 hours in the same fluid, before washing, dehydrating, etc., pre- 

 paratory to embedding in paraffin. Hematoxylin and eosin or 

 acid fuchsin were used for routine staining. Tests for the pres- 

 ence of iron were made by the potassium ferrocyanide and 

 hydrochloric acid method. 



