ESTIMATION OF OPSONIN 389 



separated from the sharp cut upper surface of the red 

 deposit of corpuscles by a narrow greyish layer of 

 leucocytes. 



5. Remove the supernatant column of citrate solution 

 by means of a teat pipette, fill normal saline solution 

 into the tube up to the upper pencil mark, and dis- 

 tribute the blood cells throughout the saline by means 

 of the teat pipette. Centrifugalise as before. 



6. Again remove the supernatant fluid and fill in a 

 fresh supply of saline solution and centrifugalis once 

 more. 



7. Remove the supernatant saline solution as nearly 

 down to the level of the leucocytes as can be safely 

 done without removing any of the leucocytes. 



8. Next distribute the leucocytes evenly throughout 

 the mass of red cells by rotating the tube between the 

 palms of the hands just as is done with a tube of 

 liquefied medium prior to pouring a plate. 



9. Set the tube upright in the plasticine block neat 

 to one end. 



Bacterial Emulsion. 



1. Take an 18- to 24-hour culture of the required 

 bacterium (e. g.-, 'Diplococcus pneumonias) grown upon 

 sloped blood agar at 37 C. Pour over the surface of 

 the medium some 5 c.c. of normal saline solution. 



2. With a platinum loop emulsify the growth from 

 the surface of the medium as evenly as possible in the 

 saline solution. 



3 . Allow the tube to stand for a few minutes so that 

 the large masses of growth may settle down ; transfer the 

 upper portion of the saline suspension to a centrifuge 

 tube and centrifugalise thoroughly. 



4. Examine a drop of the supernatant opalescent 

 emulsion microscopically to determine its freedom from 

 clumps and masses. If unsatisfactory prepare another 

 emulsion, this time scraping up the surface growth with 



