1910.] Influence of Bacterial Endotoxins on Phagocytosis. 407 



Technique. — The organisms used in these experiments were as follows : — 

 B. Ti/phosus, B. Faratyphosus, B. Achard, B. Danysz, B. Coli (several strains), 

 B. Friecllander, B. Proteus, B. Prodigiosus, B. Pyocyaneiis, Micrococcus Aureus. 

 These were cultivated on agar at 37° C, and on gelatin at 22° C, for 24 to 

 72 hours. In one instance certain of the bacteria referred to above were 

 obtained from broth flasks, which had been incubated for three weeks at 37° C. 

 The bacterial extracts were made originally from cultures grown on the 

 surface of the agar, but, for reasons which will be referred to subsequently, 

 this medium was abandoned for gelatin. 



To a sufficient growth of the organisms on plate cultivations a measured 

 quantity of sterile salt solution was added, the entire growth was carefully 

 removed, and the thick suspension of bacteria in saline transferred to a sterile 

 agate mortar. The organisms were then ground up with considerable force in 

 the presence of sterile powdered glass, or, in our later experiments, of sand. 

 The bacterial extract was then transferred to sterile tubes, centrifugalised at 

 high speed, and the supernatant fluid pipetted off into fresh tubes ; this 

 process was repeated until an extract was obtained free from bacteria. It 

 was noted that the extracts, and more particularly those obtained from 

 B. Pyocyancus and B. Proteus, were often turbid, and that the turbidity was 

 increased when the extracts were subjected to high temperatures. It was 

 undesirable in the case of certain bacteria, namely, B. Typhosus, B. Achard 

 and B. Paratyphosus, to obtain the extracts from the living organisms ; in 

 these instances the cultures were first killed by exposure to heat in the usual 

 way, but similar results were obtained whether the extract was prepared from 

 living bacteria or from those killed by heat. In a few instances the bacterial 

 suspension was ground up after being frozen in an agate mortar surrounded 

 by solid CO2. The phagocytic mixture consisted of washed human leucocytes, 

 pooled normal sera and 24 hour old suspensions in saline of living, never of 

 heated, bacteria. The whole was incubated for 15 minutes at 37° C. In 

 every case fifty leucocytes were counted with the number of bacteria engulfed, 

 and the ratio of phagocytic to non-phagocytic cells noted. 



It is convenient to mention here that extracts of organisms cultivated on 

 agar were abandoned ; because, in the majority of instances, the agar medium 

 itself was found to directly inhibit phagocytosis. We employed agar media 

 standardised to +1'5, +1'0, and +0'5 made in our laboratories, and also that 

 derived from other institutions, but similar results were obtained in all cases ; 

 when gelatin was substituted for agar the results were constant, and free from 

 error. 



An essential point in the technique, and one upon which the most 

 satisfactory results depend, is to procure abacterial extract as concentrated as 



2 K 2 



