OPSONINS— TROPINS— BACTERIAL VACCINES 187 
A second centrifiigalization is made, ami again the supernatant 
fluid, containing the last traces of blood serum, is discarded. The 
sediment, rich in leukocytes, is used as the leukocyte suspension in the 
test. 
2. Suspension of Bacteria. — Bacteria from a culture on solid media 
are suspended in sterile salt solution and agitated until a fine opales- 
cent emulsion is obtained. This is most conveniently accomplished 
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. 
Fig. 9 - Phagocytosis of strei)toc'orci 
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 de- 
scribed aboA'e. 
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 is 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 
