TECHNIQUE FOR DETERMINATION OF OPSONIC INDEX. 185 



the opsonizer (Fig. 24) and the time noted. This operation is repeated 

 with each serum. 



Coliform organisms and the gram-cocci should be incubated not longer 

 than six to eight minutes. Tubercle bacilli and other organisms require 

 fifteen minutes more or less, according to the strength of the emulsion. 



The pipettes are then withdrawn in the same order in which they were 

 placed into the opsonizer. The contents of each are blown out on to a slide 

 and very carefully mixed as before (Fig. 25). The entire quantity is divided 

 between two or three slides and several smears are made, the best one being 

 selected for counting. These slides should previously have been rough- 

 ened with very fine (oo) emery paper, cleaned with a duster, and should rest 

 on their concave surface so that the smear is made on the convex side. (It 

 will be noticed that a slide can be made to rotate if resting on one surface 

 (convex), but does not do so when resting on the concave surface). The 

 smears are best made by means of the edge of a broken slide with a slightly 

 concave edge. This "spreader" (Fig. 26) is made by sharply breaking a 



FIG. 26. 



glass slide at about its middle, this being facilitated by scratching the edges 

 of the slide with a glass cutter at the point where it is desired to break it. 

 The editor has broken as many as twenty to thirty slides before a proper 

 spreader was obtained. It pays to do this, because upon the sharpness of 

 the fracture and cleanliness of the spreader depends the edge of the film, and 

 secondarily the ease, rapidity, and accuracy of the count. If the film be 

 well made, it will have a straight edge within which will be found practically 

 all the leucocytes, as they are larger than the red blood cells, and therefore 

 dragged to the end of the film. 



The preparations are fixed in a saturated solution of corrosive sublimate 

 for two or three minutes, washed with water, and stained with methylene 

 blue or carbol-thionin (1/4 per cent, thionin, and i per cent, carbolic acid). 

 Carbol thionin is by all means preferable. It should be slightly diluted and 

 warmed before being poured upon the slide. Here it is allowed to remain 

 for several minutes, then washed off in water, and the slide dried with filter- 

 paper. The tubercle films are best fixed with formalin vapor, stained 

 with hot carbol or aniline fuchsine, decolorized in 2.5 per cent, of H 2 SO 4 , 



