1912.] 



Method for Opsonic Index Estimations. 



141 



My first problem was to get these leucocytes off the glass. This was found 

 t© be effected by citrate saline solution T5 : 08 per cent., by disodiuin 

 hydrogen phosphate, and by hypertonic salt solution, and also by serum. 

 After obtaining a large number in a test-tube in the citrate solution (to make 

 films), I found they were highly unstable osmotically and, when transferred 

 to normal saline, the majority burst. 



After experimenting with "over 200 Ponder plates, I realised that the 

 leucocytes could only be obtained in bulk if favourable chemical conditions 

 were ascertained (since incubation aggravated the bursting). The following 

 method succeeded in supplying a majority of polynuclear leucocytes (the 

 lymphocytes do not appear on the plates) in good condition, which could be 

 incubated for 15 minutes, as in the opsonic index process. The detailed 

 method is as follows : — 



Blood is shed into a rubber ring (cell) sandwiched between two glass 

 plates. This cell is incubated for 20 minutes at 37'4° C, removed from 

 incubation, the cell is opened, the clot and ring removed, the plates washed 

 with l - 25-per-cent. saline, to free them from red corpuscles and serum. 

 After wiping the ring margin clear of more red corpuscles and dried serum 

 a few drops of cold NaCl (1*25 per cent.) are poured on the leucocyte-laden 

 area of each plate. These are replaced on the metal shelf of the incubator 

 for 15 minutes ; when the plates are inspected the previously distorted and 

 stretched out polynuclears will be seen under a low objective to have become 

 almost spherical and loose from the plate. By means of a long glass rod the 

 fluid and floating leucocytes are swept into a small tube and concentrated 

 by the centrifuge at moderate speed. After syphoning off the supernatant 

 fluid a very large number of human polynuclear leucocytes were obtained, 

 which stand incubation with equal volumes of serum and the bacterial emulsion, 

 the latter being made with r25-per-eent. NaCl instead of normal saline. 



But before proceeding to test the opsonic process for the anticipated 

 increased accuracy the second defect of opportunity in the old method had to 

 be attended to. In the old method the opsonic mixture (serum, bacteria, and 

 washed blood corpuscles) was incubated for 15 minutes, but even at the end 

 of 10 minutes the bulk of the corpuscles had settled to the bottom of the 

 glass pipette, the supernatant fluid being clear. Since equality of opportunity 

 cannot exist when this is permitted this defect was remedied by keeping the 

 mixture in slow rotary motion during incubation by means of the opsonic 

 mill (fig. 1), and by this device no sedimentation occurs, but there is some 

 degree of active mixing. 



A mechanism to prevent sedimentation of the opsonic mixture was devised 

 by Bosenow, 1906, and by Glynn and Cox, 1912. 



