218 ACTIVE IMMUNIZATION 



0.5 to 1.0 mm. The ends are cut off square with a fine file. The 

 tubes are marked about 1 to 2 cm. from the ends with a glass pencil 

 and before use provided with medicine-dropper rubber nipples. One 

 volume of the leukocytic "cream" (see Preparation of Leukocytes, 

 above) is then drawn up to the mark, followed by one volume of 

 serum and one of the bacterial emulsion, the three portions being 

 separated from one another by little bubbles of air (see Fig. d, Plate 

 III). The contents of the tube are next blown out upon a slide 

 by gentle pressure upon the rubber nipple, well mixed by drawing 

 them up and down in the capillary tube, then taken up in solid 

 column, and the end sealed in the burner. The tubes are finally 

 incubated for fifteen minutes at body temperature, which may either 

 be done in an ordinary incubator or in a special "opsonifier." 



After incubation the ends of the tubes are pinched off, drops are 

 mounted upon clean slides, and after having been well mixed by 

 passage up and down in the capillary pipette, exactly in the manner 

 in which the mixture was originally made, spreads on slides are 

 prepared by the aid of the narrow edge of a second slide, as in the 

 preparation of ordinary blood smears. After drying in the air the 

 specimens may be stained with aqueous methylene blue, with some 

 polychrome dye, such as Jenner's, Hastings', Wilson's, or Giemsa's 

 stain, or with Borrell's carbol-thionin, 1 the specimens being fixed 

 with absolute methyl alcohol, if aqueous stains are to be employed, 

 while this is, of course, unnecessary in the case of alcoholic mixtures. 

 Tubercle specimens are fixed by immersion for one minute in a 

 saturated aqueous solution of mercuric chloride. They are then 

 washed off in water, stained with steaming carbol fuchsin, washed 

 with water, decolorized in 2.5 per cent, sulphuric acid, treated with 

 4 per cent, acetic acid solution to destroy the red cells, again washed 

 in water, counterstained with 1 per cent, aqueous methylene blue, 

 washed once more, and then allowed to dry. 



The average number of bacteria per leukocyte (phagocytic index) 

 is finally ascertained by going over at least a hundred cells, and the 

 opsonic index then calculated by dividing the patient's phagocytic 

 index by the normal, which is taken as unity. Example : Supposing 



1 A saturated solution of thionin in distilled water is precipitated with a 10 

 per cent, soda solution; the precipitate is collected on a small filter, washed twice 

 with distilled water, and then dissolved in 5 per cent, carbolic acid solution 

 (1 gram : 100 c.c.). The solution must always be filtered before use. 



