QUANTITATIVE ESTIMATION OF BACTERIOTROPINS 205 



4. After the final washing, the leukocytes are suspended in sufficient 

 normal salt solution until an opacity equal to a 0.3 per cent, lecithin 

 emulsion in salt solution is attained. 



Culture. Cultures should be selected with great care, in order to 

 avoid using one that displays a well-marked tendency to undergo 

 "spontaneous phagocytosis," or, on the other hand, one unduly re- 

 sistant to phagocytosis. Usually an old strain of meningococci is 

 serviceable; it is generally necessary to try out a number of strains 

 and select the best. 



Meningococci are grown for twenty-four hours on slants of glucose 

 agar. To each slant add 0.5 c.c. each of bouillon and of normal salt 

 solution, and emulsify the growth. Or the bacteria may be employed 

 in the form of a sixteen to twenty-four hour homogeneous broth culture. 

 Tubercle bacilli may either be triturated in an agate mortar with 1.5 

 per cent, salt solution added slowly drop by drop, or the tubercle powder 

 of Koch may be employed in an emulsion prepared in the same manner. 

 The resultant emulsion should be freed from coarser clumps by brief 

 centrifugalization, but, as a general rule, it is very difficult to secure a 

 uniform emulsion of tubercle bacilli by any method. 



The Test. 1. The mixtures are made preferably in a series of small 

 test-tubes about 5 cm. long and 1 cm. wide. 



2. Mix 0.1 c.c. of each dilution of immune serum with 0.1 c.c. of bac- 

 terial emulsion. Plug each tube with cotton. 



3. Mix and incubate for one hour. 



4. Add 0.1 c.c. of leukocytic emulsion to each tube. Double this 

 quantity may be used if the emulsion is weak. 



5. Mix and incubate for from one-quarter to two hours, depending 

 upon the variety of microorganism. 



6. At the end of the second incubation the leukocytes will have 

 settled to the bottom of the tubes. Carefully remove the supernatant 

 fluid from each tube; mix the sediment well with a loop, and make smears 

 on slides. Label each slide carefully to correspond to its serum dilution. 



7. Dry the smears in the air, fix with methyl alcohol, and stain with 

 carbolthionin, as previously described. 



The Controls. 1. A series of the lower dilutions of pooled normal 

 serums are set up in exactly the same manner. 



2. A tube containing bacteria and leukocytes without serum to 

 detect the extent of spontaneous phagocytosis. 



Readings. A great number of fields are examined microscopically, 

 and a note is made of the weakest dilution that still favors phagocytosis. 



