PREPARATION OF THE SERA 125 



air-bubble, and finally a similar portion of the bacterial 

 emulsion. The three droplets are carefully blown on to a slide, 

 and are thoroughly mixed with one another by being alternately 

 drawn up into the tube and expelled ten times. The mixture 

 is then drawn into the tube, and the end sealed off in the flame. 

 The rubber nipple is removed, and the tube placed in the 

 incubator at 37 for fifteen minutes. A slide is now prepared 

 by rubbing it once or twice with very fine emery paper (No. 000) 

 and thoroughly wiping it. This is a procedure adopted by 

 Wright to cause an evenly distributed film to be made. The 

 tube being removed from the incubator and the end broken off, 

 its contents are again mixed by expelling and drawing up into 

 the tube. A minute droplet is placed on the prepared slide, 

 and by means of the edge of the end of another slide a film 

 is made, which is then dried and is ready for staining. The 

 spreader should be slightly narrower than the slide on which 

 the film is made ; in this way the film has two definite edges 

 a fact of importance, as the leucocytes are usually in greatest 

 abundance near these edges. Films containing staphylococci 

 are stained either by Leishman's stain (</.v.) or with carbol- 

 thionin blue. In the former case no fixation is necessary, in 

 the latter it i.s usual to fix in saturated perchloride of mercury 

 for one and a half minutes, wash in water, and then stain. With 

 tubercle films the following is the procedure : The film is fixed for 

 twoininutfs in | erehloride of mercury, washed thoroughly, stained 

 with carbol-fuchsin as usual, decolorised with 2*5 per cent, 

 sulphuric acid, cleared with 4 per cent, acetic acid, counter- 

 stained with watery solution of methylene-blue, and dried. 



In applying the technique two preparations are made, in both 

 of which the same emulsion and the same leucocytes are em- 

 ployed ; but in one the bacteria have been exposed to the serum 

 of the infected individual under observation, and in the other 

 to that of a normal person, usually the observer himself, or 

 better still, to a mixture of sera from several normal persons. 

 Each of these preparations is now examined microscopically with 

 a movable stage, the number of bacteria in the protoplasm of at 

 least fifty polymorphonucleated leucocytes is counted, and an 

 average per leucocyte struck (this is often called the " phagocytic 

 index ") ; the proportion which this average in the case of the 

 abnormal serum bears to the average in the preparation in which 

 the healthy serum was used constitutes the opsonic index that 

 of healthy serum being reckoned as unity. 



The reliability of the opsonic method, of course, depends on 

 whether or not the phagocytic activity of the cells counted 



