OPSONIC INDEX 187 



cellular bacteriolysis explains why an individual may possess immunity 

 and yet his serum fail to show any bacteriolytic power. 



The following modification of Irishman's method takes very little time and skill 

 and is applicable in the determination of the organism concerned in an infection, as 

 in Wright's method. The control of vaccine treatment by taking opsonic indices 

 from time to time does not seem to have met with much favor in this country the 

 sources of error being as great if not greater than ordinary variations in the opsonic 

 index during the negative and positive phases. 



Method. We start with a i % solution of sodium citrate in salt solution. With 

 this emulsify a twelve- to twenty-four-hour agar slant growth of the organism to be 

 tested using 6 to 8 c.c. of the citrated salt solution. The bacterial emulsion is now 

 poured into a bottle or sealed off in a test-tube and shaken thoroughly in a shaker or 

 by hand. The emulsion is then centrifuged to throw down the bacterial clumps and 

 the supernatant slightly turbid bacterial suspension pipetted off. If working with a 

 dangerous pathogen it is advisable to kill the organisms as in making vaccines. 



Now with a capillary bulb pipette so graduated that the one volume mark con- 

 tains about o.i c.c. we draw up one volume of citrated salt solution. Then having 

 made a break with an air column, we take up one volume of the patient's blood. 

 Again make an air break and draw up one volume of the citrated salt solution 

 bacterial emulsion. The 3 volumes are then immediately forced out into a 

 small test-tube, made from 3 inches of ^ 6 -inch glass tubing, as shown in the 

 Emery technic for the Wassermann. The citrate prevents coagulation of the blood 

 and the contents of the tube are well mixed by drawing up and ejecting with the 

 capillary bulb pipette. Incubate this small test-tube at body temperature for fifteen 

 minutes, shaking the contents once or twice during the incubation period. Exactly 

 at the expiration of the period of incubation (usually fifteen minutes although at 

 times ten minutes or thirty minutes may be desirable) place the tube in a centrifuge 

 and throw down the cell sediment. Next pipette off the supernatant fluid and then 

 plunge a finely drawn out capillary pipette to the bottom of the tube and draw off 

 the greater part of the sediment at the bottom. This consists largely of the red 

 cells the leukocyte layer on the surface being undisturbed. 



Now mix the remaining cell sediment and smear out on a slide or preferably 

 between two cover-glasses as in Ehrlich's method. The smear is fixed by burn- 

 ing off a film of alcohol or with methyl alcohol and stained with dilute carbol 

 fuchsin or methylene blue. The granule staining with Wright's stain makes it 

 slightly confusing. 



A second similar preparation but using blood from a normal person as a control 

 is then made. Counting the phagocytized bacteria in a given number of poly- 

 morphonuclears, we obtain an average number of bacteria phagocytized per cell. 

 Repeating the count with the control or normal blood, we likewise have the average 

 number of bacteria taken up per cell. Dividing the patient's average by the 

 normal average, we have the opsonic index. If the average for 50 of the patient's 

 cells was 8 and that of the control only '4, the patient's index would be 2, or twice 

 the normal. The practical value of this test is that where two or more organisms 

 are on a plate from a body fluid we may ascertain the causative organism by 

 noting marked variation from the normal in the patient's opsonic index for that 



