TO DEMONSTRATE OPSONINS 319 



EXERCISE 11. TO DEMONSTRATE OPSONINS AND TO 

 DETERMINE THE OPSONIC INDEX 



Apparatus. Several .small test tubes; forty-five small 

 mixing pipettes; sterile citrated salt solution; Wright's 

 stain; suspension of leucocytes; normal serum; patient's 

 serum. 



Culture. Organism producing the disease. 



Method. 1. The small mixing pipettes are made by 

 drawing out 4 to 5 mm. glass tubing to a long, fine capil- 

 lary tube; and providing with a small rubber bulb. (Con- 

 sult the instructor for the method.) 



2. Prepare the suspension of leucocytes by collecting 

 a few cubic centimeters of blood from any animal and 

 immediately place in three or four volumes of citrated salt 

 solution. 



Centrifuge and wash at least three times, being careful 

 not to pipette off any of the cells during the washing 

 process. 



After the last washing, pipette off the supernatant 

 liquid and lay the tube in as nearly a horizontal position 

 as possible for about twenty-five to thirty minutes. At 

 this time there will appear an upper whitish layer of cells 

 composed almost exclusively of leucocytes. 



Pipette off the leucocytes. They should be used within 

 five to six hours from the time the blood is collected. 



3. Bacterial Suspension. Transfer a loopful from an 

 eighteen to twenty-four hour agar culture to 2 or 3 c.c. of 

 physiological salt solution and mix well. The suspension 

 must be carefully made to avoid clumps and some method 

 of standardization used so that successive tests will be 

 comparable. (The nephelometer may be used for this 

 purpose see an instructor.) 



4. Collect the patient's serum and normal serum at the 

 same time and under the same conditions in order that the 

 results may be comparable. The blood is collected in a 



