ESTIMATION OF THE OPSONIC CONTENT OF THE BLOOD 223 



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 bac- 

 terial emulsion. This point can only be learned by experience. For 

 convenience' sake, small glass capsules may be prepared containing 

 emulsions of barium sulphate of varying degrees of turbidity, 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 wdth normal serum the average number of organ- 

 isms 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 by exposure to sunlight for twenty-four hours. Some of the 

 material is then scraped off, ground up in an agar mortar with 1.5 per 

 cent, saline, and centrifugalized to remove clumps. Cole states that 

 if contamination is guarded against the supernatant fluid may be 

 used for at least a month. I have not had occasion to use emulsions 

 prepared in this manner, and am familiar only with emulsions made 

 from dead and ground-up bacilli. A small quantity of this material 



