126 METHODS OF EXAMINING SERUM 



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 staphylo- 

 cocci are stained either by Leishman's stain (q.v.) or with 

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

 in the latter it is usual to fix in formalin vapour for a few 

 seconds. With tubercle bacilli the following is the procedure : 

 The film is fixed, washed thoroughly, stained with carbol-fuchsin 

 as usual, decolorised with 2 '5 per cent, sulphuric acid, cleared 

 with 4 per cent, acetic acid, washed with water and counter- 

 stained with watery solution of methylene-blue (to which per 

 cent, sodium carbonate may be added), 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 a hundred polymorphonucleated leucocytes is counted, and 

 an average per leucocyte struck (this is often called the " phago- 

 cytic 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 

 represents the phagocytic activity of the cells in the preparation. 

 Considerable controversy has arisen on this point. The general 

 result may be said to be that where such organisms as the 

 pyogenic cocci are concerned, the ordinary opsonic technique 

 gives on the whole reliable results. In the case of the tubercle 

 bacillus, there is considerable difference of opinion. Generally 

 speaking, it may be said that indices varying between *8 and 



