214 PHAGOCYTOSIS. OPSONINS AND BACTERIOTROPINS 



40 to 60 c.cm. of sterile salt solution. Occasionally it may be necessary to 

 use i 1/2 per cent, sodium citrate solution instead, in order to prevent 

 coagulation. The leucocytes must be washed free of any serum. They 

 should not however be centrifugalized too rapidly, as this tends to clump 

 them. 



If rabbit's leucocytes are preferred, 50 to 100 c.cm. of 3 per cent, to 10 

 per cent, peptone bouillon should be injected intraperitoneally. -For mice 

 i c.cm. aleuronat bouillon is sufficient. Human leucocytes are obtained 

 from abscesses or from the blood (Wright). 



The serum is inactivated by heating it at 50 to 60 C. for one-half hour. 

 This may be omitted for old or carbolized sera as they are usually free of 

 complement. Also tuberculous sera should not be heated as their bac- 

 teriotropins are very susceptible to heat. 



The various serum dilutions (i : 10, i : 100, i : iooo,etc.) are prepared 

 as usual, but small quantities suffice since only 0.5 to i.o c.cm. of each dilu- 

 tion is necessary. 



The bacteria are best employed in the form of a 16 to 24 hours homo- 

 geneous broth culture. If agar cultures are used, three loopfuls are 

 rubbed up in i c.cm. of salt solution. 



For meningococci Neufeld advises agar cultures. Tubercle bacilli can either be 

 triturated in an agate mortar or bought in the form of tuberculin residuum, T. R. 

 (Hoechster Farbwerke). 



Equal parts (0.02 to o.i c.cm.) of each of the three ingredients (serum, 

 bacteria, leucocytes) are transferred by means of a capillary pipette, to 

 small tubes with flattened bottoms about 4 to 5 cm. long and i cm. wide. 

 Double the quantity of leucocytes may be needed if they are in a weak 

 suspension. 



The tubes are closed by small corks or non-absorbent cotton, gently but 

 thoroughly shaken and placed in the incubator for definite periods of 

 time, depending upon the micro-organism, from one-fourth to four hours. 

 The supernatant fluid is then pipetted off, cover-glass preparations made of 

 the sediment, fixed by heat and stained by i per cent, methylene-blue 

 solution. 



A great number of fields are examined microscopically and note made of 

 the weakest dilution which still favors phagocytosis. This is the bacterio- 

 tropic titer of the serum. 



The necessary controls are: (i) Tube containing normal serum + bac- 

 teria + leucocytes. Phagocytosis of less virulent bacteria frequently 

 occurs even with normal serum. (2) Tube containing bacteria + leuco- 

 cytes, without any serum; a virulent bacteria are sometimes taken up by 

 leucocytes even without serum. This is never the case with virulent 

 organisms. 



