PIvANT PARASITES INOCULATEJD INTO ANIMALS. 1 83 



IV. Two trout. Each two shallowpricks in the throat outside near the junction of the gill arches. 



V. Two trout. Each inoculated in the eye-socket. This time the skin was cut with a scalpel 

 and a 2 -mm. loop of the white bacterial slime was inserted. 



VI. Two trout. Each inoculated inside of the mouth at the base of the tongue by means of 

 several needle-pricks. 



VII. Four roach. Each inoculated in the eye-socket. The skin was cut and a 2-mm. loop of 

 the bacteria inserted. 



VIII. Two roach. Each received several needle-pricks in the vicinity of the anus. 



IX. Fifty salmon trout fry (2 to 3 cm. long). They were put over night into 2 liters of water 

 into which the remnant of the 4 agar cultures had been washed. The next morning they were trans- 

 ferred to running water in the ordinary shallow hatchery boxes. 



Inoculations of April 4, 1908. — For these, 5 slant agar cultures 3 days old were used. 

 The copious growth was washed off into 20 cc. of distilled water, making a milky suspension. 

 All of the inoculations were made with the hypodermic syringe. 



X. Three leopard frogs. Each received 'cc. of the very cloudy fluid. This was injected into 

 the muscles of the right thigh. 



XI. Three leopard frogs. Each received |cc. under the skin on the abdomen. In one of these 

 it was thought that the needle entered the abdominal cavity and for this reason it was kept separate, 

 but the result was not different. 



XII. Two leopard frogs and 3 green frogs (Rana clamitans). Each received jcc. in the right 

 eye-socket. 



XIII. Three brook trout. Each received 'cc. in the eye-socket. 



XIV. Three brook trout. Each received Jcc. of the very cloudy bacterial suspension. This 

 was injected into the peritoneal cavity, the needle being set in just behind the ventral fins. 



XV. The virulence of the cultures was determined by making inoculations on four young daisy 

 plants. These promptly contracted the disease. On June i these plants bore, where inoculated, 

 tumors which were over an inch long by 0.50 to 0.75 inches broad and thick. 



Results: The results so far as tumor production is concerned were either negative or 

 uncertain; all the fish have not been sectioned. The experiment was complicated by the 

 discovery after the inoculations were begun that some of the fish were suffering from car- 

 cinoma of the tongue, gills and thyroid region. Thenceforth, I examined each fish and 

 inoculated only such as appeared to be sound, but nevertheless some of them may have 

 then been about ready to develop such tumors as subsequently appeared. On March 25, 

 1908, I caught and examined 50 of the 100 check fish and found 3 with carcinomatous throat 

 tumors. Additional cases of this disease appeared in the checks, especially as the season 

 advanced. Consequently the tumors which developed on the inoculated fish may have 

 been due altogether to this disease, at least under the circumstances I could not be certain 

 that they were not so caused. 



The frogs proved very resistant. None developed tumors. Most were finally chloro- 

 formed at the end of the experiment. The few that died earlier gave no plain evidence of 

 being in any way injured by the bacteria. 



Of the roaches one died the day after inoculation. The rest died in from 17 to 32 

 days. None of the latter developed tumors. All were more or less inflamed, both roaches 

 and trout. 



The inoculated trout (except the fry which showed no signs of disease attributable 

 to the bacteria) died off faster than the checks in the main tank. They were, however, not 

 under altogether the best conditions, i.e., they were rather too crowded at the beginning 

 of the experiment and the water was several degrees too warm toward the close of the experi- 

 ment, but at the same temperature as that given to the check fish. If I were to do over 

 this experiment in a climate like that of Washington, I would begin in the autumn, so as 

 to allow the experiment to run for at least six months in cool weather. 



