OPSONIC DETERMINATIONS 261 



the proper density can be judged by experience alone, but the 

 suspension should be only faintly opalescent. 



5. Suspension of living leucocytes. To prepare this, take about 

 10 c.c. of physiological salt solution containing per cent, of 

 sodium citrate, to prevent the coagulation of the blood. This 

 must be freshly prepared (or kept sterile, which is inconvenient), 

 and the simplest method is to use the " soloids " supplied by 

 Burroughs and Wellcome ; one of these dissolved in 10 c.c. of dis- 

 tilled water will yield the solution required. This is put into a 

 centrifuge tube and warmed to blood-heat. A healthy person is 

 then pricked in the ear or finger, and his blood is allowed to drop 

 into the fluid until 1 c.c. or more has been collected. The tube 

 is then centrifuged until all the corpuscles have come to the 

 bottom and the supernatant fluid is left clear. If the deposit is 

 closely examined a delicate grey layer will be seen on the surface 

 of the red corpuscles ; this consists chiefly of leucocytes. The 

 whole of the clear fluid is then pipetted off, as close as possible to 

 the leucocyte layer, but without disturbing the latter, with a 

 pipette armed with an india-rubber teat, or with a syringe. The 

 tube is again filled with saline solution, the blood and fluid are 

 mixed, the mixture is centrifuged, and the clear fluid pipetted off, 

 and this process of washing is repeated. Next, the leucocyte 

 layer with the upper layer of red corpuscles (which also contains 

 leucocytes) is pipetted off into a small tube, and the whole is 

 thoroughly mixed by repeated aspiration into the pipette. The 

 result is a suspension of living leucocytes mixed with red corpuscles. 



The process. (1) Make a pipette and place an india-rubber teat 

 on the thick end, and with a grease pencil or with ink make a 

 transverse line about an inch from the point. 



(2) Having the patient's serum and the suspensions of leuco- 

 cytes and of bacteria ready to hand, take the pipette and suck up 

 the suspension of bacteria to the mark, i.e. one volume ; then 

 remove the point from the fluid and relax the pressure so that a 

 small bubble of air is sucked up. This will be easy if the point is 

 a good one, otherwise it will be difficult, as the column of fluid will 

 either refuse to stir or will oscillate violently. Next immerse the 

 point in the suspension of leucocytes and draw up one volume. 

 This will be separated from the suspension of bacteria by the 

 bubble of air. Kemove the point from the suspension and draw 

 up a second bubble of air. Lastly, draw up one volume of the 



