192 Transactions of the Society. 



ordinary nutrient agar, plus 10 in reaction, 10 c.cm.; tyrosin, 

 • 1 grm. ; choline, • 1 grm. The jellies were melted and poured 

 into sterile Petri dishes, 3 in. in diam., and allowed to set. Material 

 was taken in the manner already described, and was spread out on 

 the surface of the jellies. The plates were then incubated to 

 37° C. After many failures, it was found that a small amount of 

 growth took place in the tyrosiu jelly, the amoebse ultimately 

 forming cysts. Sub-cultures gave a more vigorous growth, and the 

 strain was able to be maintained. The amoebse so obtained 

 resembled those found in the pyorrhoeal pockets, with the exception 

 that a contractile vacuole was always present. Being dissatisfied 

 with the technique, improvements were tried. • 1 p.c. of creatine 

 was added to the jelly, and material was spread as before. The 

 plates were then placed in a Bulloch's apparatus, and incubated 

 anaerobically at 37° C. Working in this manner, amoebae were 

 cultivated from some 65 p.c. of the cases. The anaerobic cultures 

 were very vigorous ; the amoebae showed no contractile vacuoles if 

 examined in 0*5 p.c. saline, and indeed were absolutely indis- 

 tinguishable from those seen in mouth preparations. Moreover, 

 encystment did not take place, even if the plates were incubated 

 for three weeks. This was a very striking fact, as aerobic cultures 

 invariably encysted in four to five days, and only very scanty growth 

 took place. The cysts in this species measure on the average 

 10 to 12 /i (PI. XVI, fig. 3). In young cysts the wall may appear 

 single, but in older forms there is often a characteristic double 

 wall, with wavy irregular outline. The cysts are usually uninu- 

 cleate, but two and four nuclei have been seen. Having obtained 

 twelve different strains of this aniceba, it was considered desirable 

 to endeavour first to obtain cultures which were the progeny of a 

 single cyst, and, secondly, to grow the amoeba in pure mixed culture 

 with some definite bacterium. The first object was readily accom- 

 plished by the method described by Cropper and Drew (2). The 

 second was finally attained by a modification of this method for 

 obtaining pure mixed cultures. Four p.c. HCl was found to injure 

 the cysts, and the method finally used was to grow the material from 

 the mouth anaerobically for a week, when very numerous amoeba 

 were found. The plate was then removed from the anaerobic 

 chamber, and allowed to remain in the 37° C. incubator till numerous 

 cysts developed. Two p.c. HCl was then poured on to the plate, 

 and was allowed to act for twenty-four hours. At the end of this 

 time the acid was poured off, and as many of the cysts as could be 



EXPLANATION OP PLATE XVI. 



Fig. 1. — Entamoeba gingivalis. (Iron-hgematoxylin preparation.) 

 „ 2. — Amoeba buccalis. (Iron-haematoxylin preparation.) 

 „ 3. — Cysts of A. buccalis, young and old forms. (Iron-hsematoxylin stain.) 



