TECHNIC 



197 



tubes centrifuged. Slight discoloration of the serum from mechanically 

 breaking-up a few erythrocytes will not interfere with the test. 



4. If gross contamination is avoided, blood may be kept for one or 

 two days in a dark place without much deterioration of its opsonin 

 content. 



5. It is always well to collect the control bloods at the same time the 

 patient's blood is taken, or, if this cannot be done, to place them in an 

 ice-chest as soon after collection as possible. When conducting the 

 test, the control serums are pooled and mixed in a clean watch-glass. 



The Bacterial Emulsion. 1. This must be perfectly uniform, free 

 from clumps, and must not undergo agglutination, either spontaneous 

 or with the serum to be tested. 



With many bacteria, especially the motile ones, such as Bacillus 

 coli and Bacillus typhosus, it is comparatively easy to secure a uniform 

 emulsion. Staphylococci, streptococci, and pneumococci, as a rule, 

 present no difficulties. After growing the culture for from eighteen to 

 twenty-four hours on slants of a suitable medium, add three cubic centi- 

 meters of sterile 1 per cent, salt solution, and gently remove the bac- 

 terial growth with a platinum loop. The mixture is then pipeted into a 

 separate flask or thick glass test-tube containing glass beads, and shaken 

 by machine or by hand until it is thoroughly emulsified. If necessary, 

 the emulsion may be centrifugalized to remove clumps and is then ready 

 for use. 



2. It must consist of bacteria that stain evenly and well. 



Only young cultures should be used; for example, an eighteen to 

 twenty-four-hour culture of freely growing organisms and a seven days' 

 growth of tubercle bacillus. 



3. It must be of such strength as to give a leukocytic ingest that will 

 enable adequate differentiation to be made of the opsonin content of the 

 various serums. 



An emulsion that does not yield a count of at least 250 bacteria 

 within 100 leukocytes is too weak to yield a satisfactory differential 

 count. If the emulsion is too thick, bacteria overlie the leukocytes 

 and introduce error. As a general rule, a suspension containing 500,- 

 000,000 bacteria per cubic centimeter is satisfactory. Experience will 

 teach the right density to be used, and frequent trials may be necessary 

 before the right one is secured. 



4. The bacteria must be such as will not prove seriously dangerous. 

 In order to obviate any danger attending work with such organisms as 

 the glanders and the tubercle bacillus, first kill the culture by pouring 



