TECHNIC 195 



on a 10 to 40 per cent, solution of formalin, mix the culture, shake, 

 transfer to a centrifuge tube, and centrifugalize until the bacteria have 

 been carried to the bottom of the tube. Pipet off the supernatant 

 formalin, wash in normal salt solution, centrifuge, pipet off again, and 

 finally mix the sediment in sufficient salt solution to make a satisfactory 

 suspension. 



The Washed Leukocytes. These should consist mainly of the poly- 

 nuclear leukocytes of a healthy person washed free from any admixture 

 with serum. As usually obtained, the leukocytes are mixed with red 

 corpuscles. It is necessary to collect blood for the leukocytic mixture 

 from a person whose corpuscles are known to be insensitive to agglutina- 

 tion, as otherwise there is an undue lowering of the opsonic effect. 



1. Place 4 c.c. of sterile 2 per cent, solution of sodium citrate in dis- 

 tilled water in sterile 10 c.c. centrifuge tubes. 



2. Prick the finger, and add 1 c.c. of blood. Agitate well to insure 

 thorough mixing. 



3. Centrifuge at a sufficiently high speed to mix the red corpuscles 

 and leukocytes at the bottom of the tube and avoid clumping of the 

 leukocytes. 



4. Draw off the supernatant fluid, add 5 c.c. of 1 per cent, salt solu- 

 tion, mix, and centrifuge. 



5. Wash once more. Draw off the supernatant fluid. 



6. Add sufficient salt solution to bring the total volume up to that of 

 the blood originally taken i. e., 1 c.c. Mix well. 



The Test. 1. Prepare capillary pipets of approximately the same 

 caliber. These are made by taking 6-inch lengths of soft clean glass 

 tubing having an external diameter of -f$ inch, heating them in the 

 middle in the tip of the blow-pipe or the Bunsen flame until about J^ 

 inch length of tubing is quite soft. Remove from the flame, and by rap- 

 idly separating the two hands draw out the molten glass to a length of 

 from 18 to 20 inches. After cooling, the capillary thread is cut across 

 with a small file, so that from 6 to 8 inches is left attached to each piece 

 of tubing. The ends must be cut square, as ragged and uneven ends 

 are difficult to handle. By means of a wax-pencil make a fine mark at a 

 point about an inch from the free end of each capillary thread. This 

 indicates the unit volume (Fig. 48). 



2. Adjust a well-fitting rubber teat, and draw up the unit volume of 

 blood-cells. A tiny bubble of air is now allowed to enter the thread, and 

 then one volume of the bacterial emulsion is added; another air-bubble 

 is allowed to enter, and finally one volume of serum, so that we have 



