SMEARS. 33 



limit the longer the period of incubation the more marked will 

 be the phagocytosis. The incubation time must in some cases 

 be limited because of solution and agglutination of bacteria. 

 In all instances the mixture containing patient's serum and 

 that containing normal serum must be incubated at the same 

 temperature and for the same length of time. The time of in- 

 cubation varies from five to thirty minutes, depending upon the 

 species of microorganism and the properties of the blood serum 

 tested. 



After incubation the nipple is removed and the capillary 

 end of the pipette is broken off, the nipple is then replaced and 

 the leucocytes, bacteria and serum are again thoroughly mixed 

 on a glass slide, after which smears are made. 



SMEARS. 



If reference again is made to the principle involved in the 

 determination of the opsonic index, it will be noted that the index 

 shows the ratio of the number of bacteria taken up by the poly- 

 morphonuclear neutrophiles when patient's serum is a part of 

 the mixture, to the number of bacteria taken up by the same 

 class of leucocytes when normal serum is a part of the mixture. 

 It is therefore necessary to count the number of bacteria in the 

 polynuclear leucocytes. In order to have an abundance of leuco- 

 cytes the grayish red layer is taken off the centrifuged blood. 

 This contains a relatively larger number of white blood cells than 

 does blood freshly drawn. If a drop of this leucocytic cream 

 is spread out by dropping a cover glass onto it, fixed, and stained, 

 many red blood cells and some white blood cells will be found in 

 the field of the microscope. It would, however, be a tedious 

 task to find fifty or one hundred polynuclear neutrophiles. Wright 

 and Douglas have devised an ingenious method to gather the leuco- 

 cytes together in certain parts of the slide. The method of doing 

 this is based on the difference in size of red blood cells, mononu- 

 clear and polynuclear leucocytes; the polynuclear and large mon- 

 onuclear cells being largest in size. 



The method of Wright and Douglas is as follows: A slide is 

 cleaned and slightly scratched by rubbing with jeweler's emory 

 paper to slightly roughen the surface of the glass slide. On the 

 left end of this slide a drop of the incubated mixture, which has 



