OPSONINS 831 



(a) Describe the exudate. How does it differ from that found in 

 the preceding experiment? 



(b) How do you explain the serous character of the exudate? 



(c) Has phagocytosis occurred? 



(d) Do you think the streptococci have multiplied in the peritoneal 

 cavity? 



(e) What means could you suggest for overcoming this action of 

 virulent streptococci? 



EXERCISE J2. OPSONINS 



EXPERIMENT 35. NORMAL OPSONINS 



1. Prick the finger and secure 1 c.c. of blood in a small test-tube. Also 1 c.c. in a 

 centrifuge tube containing 2 c.c. of sodium citrate solution. After coagulation re- 

 move the serum from the first tube. 



2. Divide the serum into two portions and heat one portion at 56 C. for thirty 

 minutes. 



3. Prepare an emulsion of leukocytes by centrifuging the blood collected in the 

 citrate solution, removing the supernatant fluid, adding normal salt solution, and 

 centrifuging again. Repeat this step once more in order to wash the cells thoroughly 

 and after the last centrifuging remove the supernatant fluid and add sufficient salt 

 solution to make the total volume 1 c.c. and mix thoroughly. 



4. Prepare an emulsion of staphylococci which is homogeneous and free of clumps. 



5. Mark two capillary tubes with a wax pencil about an inch from the tip; fit 

 rubber teats to the other end. 



6. With pipet No. 1 take up a volume of blood-cells; allow a bubble of air to 

 enter and then an equal volume of bacterial emulsion; bubble of air and an equal 

 volume of the fresh unheated serum. Mix well by alternate expulsion and aspiration 

 on a clean slide. Then draw the whole into the stem of the pipet and seal the tip in 

 a flame. 



7. Repeat with pipet No. 2, using the heated serum. 



8. Incubate both pipets at 37 C. for half an hour. 



9. Remove the pipets from the incubator, break off the tips, mix the contents, 

 and prepare smears. 



10. Fix the smears with a saturated solution of bichlorid of mercury for one 

 minute; wash in water and stain with carbol-thionin for two minutes; wash in water 

 and dry. 



11. Examine with oil-immersion lens. 



(a) What are the requisites of a satisfactory opsonic preparation? 



(b) Is there any difference in the amount of phagocytosis between 

 the heated and unheated serums? If so, how do you explain the result? 



(c) Do normal opsonins play any role in natural immunity? 



(d) Give the properties of normal opsonins. 



