RESEARCHES ON AVIAN DIPHTHERIA. 493 



ism obtained was the cause of an accessory infection, and simply 

 associated with, but not the specific cause of, the disease. 



It has seemed to us, therefore, necessary on account of the ten- 

 dency of the disease to remain localized, to isolate the organism 

 from the morbid product itself rather than attempt a purification 

 of culture in the animal body. A microscopical examination 

 of a bit of false membrane sufficed, however, to convince us that 

 this product is very unsuitable for purposes of direct isolation. 

 The membrane, indeed, is swarming with different bacteria, the 

 separation of which would be a difficult and probably fruitless 

 task. The buccal cavity contains, as we know, under normal 

 conditions, many bacterial species, and these ordinary organisms 

 are still more increased when lesions are present. It seemed better, 

 then, to produce the specific infection in some region of the body 

 which is better protected from saprophytes, and the nictitating 

 membrane in the hen's eye seems to answer this requirement. A 

 thread that has been wet in water in which a fragment of false 

 membrane has been suspended is passed through the nictitating 

 membrane. On the following day this thread is withdrawn, and 

 the irritation due to trauma disappears. Three or four days later 

 a grayish point on the nictitating membrane appears at the point 

 of inoculation. 



This point then becomes red and thickened, and soon shows a 

 typical diphtheritic lesion; a marked edema is also noticeable about 

 the eye. At this time, that is, about the eighth day, the nictitating 

 membrane is cut out and washed in sterile salt solution, and then 

 ground up in a few drops of the same solution. The suspension 

 obtained in this way is inoculated on the glycerinated-potato-blood- 

 agar medium which Gengou and I have employed in growing the 

 whooping-cough bacillus. This suspension when inoculated on 

 the buccal membrane of a normal hen produces typical diphtheria. 

 The surprising thing is that microscopical examination with a 

 stain of carbolated toluidin blue or with the Giemsa stain shows 

 no definite bacteria; a few granulations, and at times somewhat 

 elongated spots, are seen, which are so small as not to be distin- 

 guishable from cell debris. The culture tubes inoculated with this 

 emulsion are incubated for 3 or 4 days. Only a very small num- 

 ber of colonies is evident after this period; and these colonies 



