

OPSONINS 241 



4. After washing three times decant the supernatant liquid being careful 

 not to stir up the deposit of cells. Lay the tube as nearly horizontally as 

 possible and leave it for about half an hour. Then collect the upper whitish 

 layer of cells which is composed almost exclusively of leucocytes. The leuco- 

 cytes ought to be used within 6 hours of the blood being collected (Milhit). 



Experimental details. 



1. Take a Pasteur pipette ready furnished with an india-rubber teat, cut 

 off the capillary end squarely with a carburundum pencil and make a small 

 mark on the glass about 2 cm. from the point. 



FIG. 162. Preparation of the mixture for the determination of the opsonic 

 index. The figure shows the three equal volumes of fluid aspirated into the 

 pipette and separated by two bubbles of air. 



2. Aspirate into the pipette in turn by lightly relaxing the teat : 



(a) A column of leucocytes up to the mark on the glass, then a small 

 bubble of air. 



(6) A column of bacterial emulsion up to the same mark, then another 

 bubble of air. 



(c) A column of the serum to be examined, again up to the mark. 



There are now three equal volumes of fluid in the pipette separated by two 

 small bubbles of air (fig. 162). 



3. Expel the liquids on to a sterile slide and mix them together, then 

 draw up the mixture into the pipette again, being careful to avoid taking up 

 any air bubbles. Seal the end in the pilot of a Bunsen, and place the pipette 

 horizontally in the opsonic incubator at 37 C. for 15 minutes. 



4. Break off the end of the pipette. Place a drop of the mixture on each 

 of several slides, spread rapidly, dry and fix the films by heat or alcohol- 

 ether. Stain with carbol-thionin or in the case of the tubercle bacillus with 

 carbol-fuchsin. 



5. With an oil-immersion lens count the number of the organisms phagocy ted 

 by 50 leucocytes. 



For example, 120 organisms are counted in 50 leucocytes : the opsonic 

 power of the serum examined is therefore --?- = 2*4:0. 



6. Repeat the experiment using normal serum. Suppose 90 organisms 

 are counted in 50 leucocytes : the opsonic power of the normal serum is 



o _ 1 .of\ 

 5(7 1 OU. 



(For clearness of description the two investigations the opsonic power of 

 the suspected serum and that of the normal serum have been described 

 successively. In practice, of course, they will be taken in hand together.) 



7. The opsonic index being the ratio of the opsonic power of the suspected 

 serum to that of the normal serum is : 



2-40 _ 1.QO 

 1-80 ~ 1 * 



