ESTIMATION OF THE OPSONIC CONTENT OF THE BLOOD 209 



rhage is prevented. After twenty hours the pleural cavity will 

 contain an exudate rich in leukocytes, which is pipetted off, placed 

 in citrate-saline, and washed as described above. 



Ordinarily the leukocytes should not be kept longer than five or 

 six hours. 



Preparation of Bacterial Emulsion. As the Wright technique neces- 

 sitates working with uniform emulsions, i. e., with emulsions in 

 which the bacteria are evenly distributed, this step is really the crux 

 of the whole process. With certain organisms, such as the staphy- 

 lococci, the difficulty is not so great, but with others, notably the 

 tubercle bacillus, it is almost impossible to obtain uniform results. 



Staphylococci and streptococci may be grown on plain agar, while 

 gonococci, pneumococci, and meningococci are cultivated on blood 

 agar or hydrocele agar. Small tubes, like the one pictured at b (Plate 

 III) are charged with a little saline (0.85 to 1.2 per cent.). A bit of 

 the culture is removed with a platinum loop and gently rubbed 

 against the wall of the tube, at the surface of the liquid, until a 

 uniform turbidity results throughout the specimen. This is then 

 centrifugalized for a minute or two, so as to remove clumps as 

 far as possible, and to obtain the desired degree of density of the 

 bacterial emulsion. This point can only be learned by experience. 

 For convenience sake, small glass capsules may be prepared con- 

 taining emulsions of barium sulphate of varying degrees of turbid- 

 ity, and corresponding to bacterial emulsions of standard strength. 

 With these the centrifugalized specimen may be compared before 

 use in the actual experiment. Wright advocates an emulsion of 

 cocci of such strength that with normal serum the average number 

 of organisms per leukocyte (see below) is about four or five. 



It has been recommended that the cultures should not be more 

 than twenty-four hours' old. This, however, is not necessary for 

 all organisms. Knorr has shown in my laboratory that the same 

 degree of phagocytosis is obtained with cultures of the staphylococcus 

 more than a month old, as with young cultures. In the case of 

 the typhoid and the colon bacillus, Wright recommends the use 

 of cultures only four hours' old, as with older cultures the resultant 

 spherulation of the organisms is such that approximative results 

 only can be obtained. 



In the case of the tubercle bacillus, Cole obtained the best results by 

 starting with living cultures on glycerin agar, which had been killed 

 14 



