CELLULAR RESISTANCE 159 



those which absolutely require the intervention of an opsonin 

 and those mentioned above, which apparently need little or no par- 

 ticular opsonization. 



Experimental Demonstration. For the experimental demonstration of 

 opsonization it is necessary to have washed leucocytes, bacterial suspension and 

 blood serum. Large quantities of leucocytes may be obtained by injecting 5.0 

 c.c. aleuronat suspension into a guinea-pig's peritoneum and withdrawing the 

 exudate at the end of twelve to twenty-four hours. These may be suspended 

 in five to ten volumes normal saline, gently mixed and centrifuged, the process 

 being carried out three times, when the cells are said to have been washed three 

 times. If human leucocytes are desired, 10.0 c.c. saline sodium citrate are placed 

 in a centrifuge tube and 2.0 c.c. blood added. The tube is centrifuged at high 

 speed, whereupon a layer or " cream " of leucocytes collects at the upper level 

 of the cell mass. The cream can be removed by a drawn-out nipple pipette 

 and the cells washed as indicated for the peritoneal exudate. The bacterial 

 emulsion can be made from a twenty-four-hour slant agar culture of staphylo- 

 coccus pyogenes aureus by adding 10.0 c.c. salt solution, allowing to stand for 

 ten to fifteen minutes and then rotating the tube between the palms of the hands. 

 This is pipetted into another tube and for safety may be killed by heating in a 

 water bath at 55-6o C. for two hours. The serum may be obtained by allowing 

 blood to clot and then drawing off the serum. Small quantities may be obtained 

 by the use of a tube such as shown in Fig. 8. Having these ready, 0.5 c.c. 

 bacterial emulsion are mixed with o.i c.c. serum and incubated for one-half 

 hour, then washed three times and the organisms resuspended in 0.5 c.c. saline. 

 Several capillary pipettes are made from glass tubing (5 mm. bore) and the 

 upper end flanged so as to take a rubber nipple. A mark is made with a grease 

 pencil about 2 cm. from the tip, which serves as a volume indicator. (Fig. n.) In 

 the experiment one volume bacterial suspension, one volume bacterial emulsion and 

 one volume serum or saline are drawn into the pipette each in succession to 

 the mark, permitting a small amount of air to enter before the next volume is 

 taken up so as to permit of exact measurement of the volume. These are blown 

 into a watch crystal and mixed by blowing in and out several times ; then taken 

 into the capillary again and the end sealed. After incubation at 37 C. for 

 fifteen minutes the tip is broken, the mixture dropped on slides or cover slips, 

 spread, dried and stained with Wright's stain or some other modification of 

 the Romanowsky stain. Then the number of bacteria in a given number of 

 leucocytes (20 to 50 or more) are counted and the average calculated. A sample 

 protocol follows : 



Average 



phagocytosis 



per cell 



1. Leucocytes (washed) -f bacteria (untreated) -f serum 22 



2. Leucocytes (washed) -j- bacteria (untreated) -j- NaCl I 



3. Leucocytes (washed) -j- bacteria (treated) -f- NaCl 14 



In the above protocol it is seen that the leucocytes exhibit a slight capacity 

 for taking up bacteria independently of the presence of serum, but that this is 

 much augmented either in the presence of serum or by previously treating the 

 bacteria with serum. 



Normal Opsonins. As has been indicated, the phagocytosis of bac- 

 teria and other cells is greater in immune than in normal animals, the 

 difference being due to increase in the opsonin content of the serum 

 of the immune animal. It was soon observed that the opsonin of the 

 serum of normal animals could be destroyed by heat to 60 to 65 C. 

 for 10 to 15 minutes; whereas the opsonin of immune animals is not 

 destroyed by heat of 62 to 63 C. for forty-five minutes. Similarly 

 exposure to light at room temperature leads to deterioration of normal 

 opsonin in a few days but has practically no effect on immune opsonin. 

 These differences in behavior were at first thought to constitute an 



