PHAGOCYTOSIS AND OPSONINS 127 



Have ready, 4 small centrifuge tubes of about 10 c.c. capacity each ; 

 1 solid watch-glass and small glass rod ; 

 an agar-tube culture of the bacteria, e.g. staphylococcus ; 

 1 glass tube drawn to coarse capillary size at one end ; 

 1 glass tube with rubber teat at one end and drawn out to a 



capillary size at the other ; 

 a mixture of normal saline with 1 per cent, sodium citrate, equal 



parts ; 

 microscope with oil-immersion objective, some microscopic slides 



and cover-glasses, glass vessel with methyl alcohol, and some 



* Leishman stain ' ; 

 hand centrifuge. 

 Access to an incubator, at 37° C, is requisite. 



III. In the decapitate preparation provided expose a femoral artery, 

 insert a cannula into it (exerc. XI) and draw 5 c.c. blood into two of the 

 centrifuge tubes A and B, Set them to stand so that the blood may clot. 



In the two other centrifuge tubes C and D place 5 c.c. of a mixture of 

 equal parts of 1 per cent, sodium citrate solution (to prevent clotting) and -9 per 

 cent. NaCl. Bleed into each of these about -25 c.c. of blood from the femoral. 



IV. Centrifuge the tubes C and D, placing them opposite to each other in Obs. 94. 

 the. centrifuge so as to balance. After centrifuging, the leucocytes will be seen ' Washed ' 

 as a faint white disk-like layer above the red corpuscles. Draw ofp by the 



coarse capillary pipette the supernatant fluid as far as you can without 

 disturbing the corpuscles. Add some of the sod. citr. and normal sal. 

 solution so as to half fill the tube again, and mix up the corpuscles with 

 it, avoiding frothing. Centrifuge again. Draw off supernatant fluid as 

 before, and repeat dilution and centrifuging once more. The leucocytes 

 are thus practically freed from the plasma of their blood, i. e. are 

 ' washed '. 



Y. Obtain an emulsion of the bacteria by scraping off a little of the 

 culture-growth from the agar, and mixing the scraping thoroughly with some 

 normal saline in the solid watch-glass. Any naked-eye clumps of bacteria 

 may be removed by centrifuging the emulsion mixture. 



YI. Centrifuge the clotted blood in the two tubes A and B you set 

 to stand. After centrifuging, the serum is seen as the supernatant fluid. 

 Draw off the serum from B into a small test-tube, heat it for 10' at 60° C, and 

 then allow to cool. 



