168 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS 



in a shaking machine, but repeated shaking in a stoppered test-tube 

 containing glass beads will usually suffice. The coarser clumps of 

 bacteria are removed by filtration through a coarse filter paper. The 

 density of the bacterial suspension should be such that not more than 

 ten bacteria per leukocyte will be taken up as the average. 



3. Serum. (a) Blood from three or four normal individuals is 

 collected in capillary tubes; after the serum has separated a "pool" 

 or mixture is made, composed of equal volumes of each serum. Experi- 

 ence has shown that "pooled" serum furnishes a more reliable normal 

 opsonic index than that obtained from a single individual. 



(6) Serum from the Patient. This is prepared in the manner 

 described above. 



The Test. A capillary pipette of 1 to 1.5 mm. bore is made by 

 drawing out a piece of glass tubing previously softened in the flame. 

 If the tubing is heated in the center until it softens, then, after a few 

 seconds, drawn slowly and steadily out, the desired size and shape is 

 readily obtained. A close-fitting rubber bulb attached to the larger 

 end is a convenience. 



A mark about 1 to 1.5 cm. from the capillary end is made with a 

 wax pencil, and a volume each of the leukocytes, pooled serum, and 

 bacterial suspension are drawn into the pipette. It is convenient to 

 separate each ingredient by a small air bubble, to insure uniformity 

 of volume. Mixing is accomplished by carefully expelling and drawing 

 back the respective elements into the pipette. Finally, the mixture 

 is drawn well up into the pipette, the end is sealed in the flame of a 

 Bunsen burner, and the charged pipette is placed in the incubator 

 at 37 C. This is the normal or control. 



A precisely similar preparation is made, using the serum of the 

 patient in place of the pooled serum. 



Incubation is maintained for fifteen minutes. 



The ends of the pipettes are now broken off, and the contents of 

 each pipette mixed as before. A large drop of each respective mixture 

 is spread upon clean glass slides, using the same technic as that for 

 preparing a blood smear, and air-dried. The preparations are stained 

 with Loffler's methylene blue, Wright's stain, or other stain suitable 

 for the organism used. 



The number of bacteria in fifty, one hundred, or two hundred leuko- 

 cytes are determined by direct count, and the average number of 

 bacteria per leukocyte of the normal serum compared with the average 

 number of bacteria per leukocyte in the pathological serum: 



