OPSONINS 909 



No. 3: Equal parts of contents of tube 3 and bacterial emulsion. 

 No. 4: Equal parts of contents of tube 4 and leukocytic mixture. 

 10. Incubate both pipets for fifteen minutes, prepare smears, and stain with car- 

 bol-thionin. 



(a) Carefully compare the degree and extent of phagocytosis in the 

 four mixtures. 



(b) Has leukocytosis occurred in mixture No. 2 ? If so, what special 

 term is applied to phagocytosis in the absence of serum? 



(c) What role does serum play in phagocytosis? Explain the 

 mechanism involved. 



(d) Discuss the question of "stimulin" and opsonin as demonstrated 

 by the results in preparations Nos. 3 and 4. 



EXPERIMENT 39. SPECIFICITY OF OPSONINS 



1. Secure 1 c.c. of the serum of a rabbit immunized with staphylococci and heat 

 at 56 C. for thirty minutes. Divide into two portions in separate small test-tubes. 



2. To No. 1 add 1 c.c. of normal salt solution and 4 or 5 loopfuls of a twenty- 

 four-hour agar slant culture of staphylococci. Incubate for thirty minutes; centri- 

 fuge thoroughly and remove the supernatant fluid (diluted serum) to a separate tube. 

 Call this "treated" serum. 



3. To the untreated serum add 1 c.c. salt solution, so that both are diluted 

 equally. 



4. Prepare an emulsion of rabbit or human leukocytes. 



5. Prepare an emulsion of staphylococci, homogeneous and free of clumps. 



6. Prepare two opsonic mixtures: No. 1 containing equal parts of blood suspen- 

 sion, bacterial emulsion, and untreated serum; No. 2 containing equal parts of blood, 

 bacterial emulsion, and treated serum. 



7. Incubate for thirty minutes. During this interval proceed as follows: 



8. Prepare an emulsion of typhoid bacilli, homogeneous and free of clumps. 



9. Secure 0.5 c.c. of serum from a rabbit immunized with typhoid bacilli and 

 heat at 36 C. for thirty minutes. 



10. Prepare the following mixtures in capillary pipets: 



No. 3 blood-cells + typhoid serum -|- typhoid bacterial emulsion. 



No. 4 blood-cells + typhoid serum +staphylococcus bacterial emulsion. 



No. 5 blood-cells +staphylococcus serum -{-typhoid emulsion. 



11. Incubate for fifteen minutes. 



12. Prepare smears of all and stain with carbol-thionin. 



(a) Is there any difference in the degree of phagocytosis in mixtures 

 No. 1 and 2? If so, why? 



(b) Are opsonins specific? 



(c) Has phagocytosis occurred in the cross-mixtures of staphylococci 

 with typhoid serum, and typhoid bacilli with staphylococcus serum? If 

 so, how do you explain this apparent lack of specificity? 



(d) What may happen in a mixture of fresh unheated typhoid serum, 

 typhoid bacilli, and leukocytic emulsion? 



