10 



outline and a lobed and wrinkled appearance to the surface, the color 

 being a light, clear orange. When growth is strong the colonies pile 

 up in pronounced yellow, viscid drops." In plate cultures of neutral 

 gelatin the colonies consist after a time of a few light-colored zoogloea, 

 "with a surrounding irregular area of actively growing bacteria, the 

 whole having a light cream colo)'." The germ liquefies gelatin slowly. 

 The formation of zoogloea is also particularly marked in fluid culture 

 media, such as a broth made of corn seedlings or of potato tubers. 

 The germ stains readily in all stages of development with most of the 

 dyes in general use, especially aqueous solution of fiichsin and of gen- 

 tian violet. 



DIFFICULTIES MET WITH IN ISOLATING THE ORGANISM. 



As before stated, Arthur and Bolley found more or less difficulty in 

 isolating the germ from the diseased tissues, even the acidifying of the 

 gelatin only partially preventing rapidly growing organisms from over- 

 running the cultures. Attempts to free the surface of the leaves from 

 other germs by washing with corrosive sublimate (1:1,000 solution) 

 always gave negative results: They believed that "some of the poison 

 passed over into the culture and prevented growth, even when the leaf 

 was well washed with distilled water after its treatment with corrosive 

 sublimate. Most of the work was done with cultures obtained b}^ 

 passing the leaf quickly through a Bunsen gas flame two or three 

 times, thus destroying all surface molds and bacteria in both spore and 

 vegetative condition, and then cutting the leaf into thin sections with 

 flamed scissors, allowing the sections to drop into the nutrient medium." 



The first point to be investigated was the adequacy of this method 

 -of surface sterilization. Numerous experiments b}^ the writer have 

 shown conclusivel}^ that unless the epidermal cells of the carnation are 

 killed by the heat the organisms lodged in crevices of the cuticle are 

 not always killed, l)ut often grow readily when a portion of the flamed 

 though still living epidermis is put into nutrient media. Flaming the 

 leaf, therefore, in the manner described is sufficient to remove from it 

 only the most exposed and least resistant organisms. 



It is to l)e regretted that Arthur and Bolley did not describe the poured 

 plate cultures made direct from the diseased tissues, as this is a nmch 

 more accurate method of research than simply dropping fragments of 

 tissue into tubes of culture media and then making poured plates from 

 the growth thus obtained. If the diseased mesophyll cells contain even 

 a few germs, hundreds of colonies ought to be obtained by breaking up 

 these cells and making poured plates from them direct. If onlj^ a few 

 colonies develop after the cells are broken up in quantity and such 

 plates made, the direct evidence that the germ had anything to do 

 with causing the disease would be very inconclusive, especially if the 



