340 INFECTION AND IMMUNITY 



Emulsions of tubercle bacilli are more difficult to make. The 

 bacilli filtered off in the manufacture of old tuberculin are com- 

 monly used. These are washed in salt solution on the filter, and 

 are then scraped off and sterilized. They are then, in a moist 

 condition, placed in a mortar and thoroughly ground into a paste. 

 While grinding, salt solution 1.5 cent) is gradually added until a 

 thick emulsion appears. This emulsion may be diluted and larger 

 clumps separated by centrifugalization. 



(3) The leucocytes are obtained by bleeding from the ear or 

 finger directly into a solution containing eighty-five hundredths per 

 cent to one per cent of sodium chlorid and five-tenths to one and 

 five-tenths per cent of sodium citrate. Ten or fifteen drops of blood 

 to 5 or 6 c.c. of the solution will furnish sufficient leucocytes for a 

 dozen tests. This mixture is then centrifugalized at moderate speed 

 for five to six minutes. At the end of this time, the corpuscles at 

 the bottom of the tube will be covered by a thin grayish pellicle, 



FIG. 42. PIPETTE WITH THREE SUBSTANCES, CORPUSCLES, BACTERIA AND SERUM, 



AS FIRST TAKEN UP. 



the buffy coat, consisting chiefly of leucocytes. These are pipetted 

 off with a capillary pipette (by careful superficial scratching move- 

 ments over the surface of the buffy coat). 



There being, of course, no absolute scale for phagocytosis, when- 

 ever an opsonin determination is made upon an unknown serum, 

 a parallel control test must be made upon a normal serum. This 

 normal is best obtained by a "pool" or mixture of the sera of five 

 or six supposedly normal individuals. 



The three ingredients serum, bacterial emulsion, and leucocytes 

 having thus been prepared, the actual test is carried out as follows : 

 Capillary pipettes of about six or seven inches in length and of 

 nearly even diameter throughout, are made. These are fitted with 

 a nipple and a mark is made upon them with a grease-pencil about 

 2 to 3 cm. from the end (Fig. 42). Corpuscles, bacteria, and serum 

 arc then successively, in the order named, sucked into the pipette 

 up to the mark, being separated from each other by small air- 

 bubbles. Equal quantities of each having thus been secured, they 

 are mixed thoroughly by repeatedly drawing them in and out of 

 the pipette upon a slide. The mixture is then drawn into the pipette ; 



