22 CROWN-GALL OF PLANTS. 



ever}'- appearance of being a saprophyte. Moreover, when the galls 

 were placed under favorable conditions for the development of 

 fungi which might possibly have been overlooked in the micro- 

 scopic examination, no fungus appeared. 



Bacteria in the interior of the undecayed galls were first detected 

 by the senior writer in some fresh unstained thin sections which had 

 been prepared by Dr. Townsend. Wliether these were actually 

 the bacteria we have since isolated is uncertain. These bacteria 

 occurred sparingly in small clumps but were so unmistakable, when 

 once actually seen, that it was agreed forthwith to make the disease 

 a subject of further study. With this end in view Miss Alice C. 

 Haskins, who had been trained in the senior writer's laboratory and 

 who was then an assistant in Dr. Townsend's laboratory, was 

 directed to make agar-poured plates from the interior of suitable 

 galls; and, with the bacteria so obtained, inoculations on healthy 

 daisy plants. This work proceeded for many months without pos- 

 itive results. Bacteria of several sorts were obtained frequently 

 from the interior of the galls, sometimes in abundance, but all the 

 inoculations were negative. Several factors contributed to this 

 result. It was not then known that the true parasite comes up 

 slowly on agar-poured plates (three to six days or more being required) , 

 nor that the tissues frequently contain a variety of saprophytes which 

 develop rapidly on agar. Probably most or all of the inoculations 

 of this period were made with saprophytic organisms, i. e., with those 

 first appearing on the poured plates. 



In studying the relation of bacteria to these galls, we found little 

 encouragement in the microtome sections, either stained or unstained. 

 The stain used in this connection was carbol fuchsin, with the result 

 that wdth high powers granules could be seen in and around some of 

 the cells, but these were few in number and did not seem to have the 

 characteristic even outline of bacteria. This material was fixed in 

 alcohol. 



The cultural methods used by Miss Haskins were as follows: Galls 

 were crushed in beef broth, from which agar-plate cultures were 

 made. For this purpose, fresh, soft galls of small size were used, as 

 well as galls of larger size and firmer texture. In preparing the galls 

 for these cultures, the common technic of the laboratory was used, 

 i. e., the surfaces were scraped off with a sterile scalpel; the galls 

 were then washed for 30 seconds in mercuric chlorid water (1 : 1,000), 

 and then in sterile water. After this they were cut into small pieces 

 with a sterile knife and placed in tubes containing 10 c. c. of neutral 

 sterile peptonized beef broth, one tube being used for each gall. The 

 galls were then crushed as much as possible with a sterile glass rod. 



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