DISCOVERY OF THE BACTERIA. 23 



At intervals of two to four weeks during the greater part of two 

 years agar plates were poured from preparations made in this manner. 

 These plate cultures, which altogether amounted to several hundred, 

 were kept at temperatures varying from 20° to 30° C. Both white 

 and yellow colonies of different shapes and tints appeared from time 

 to time, and some of the plates seemed to contain pure cultures, but 

 none of the organisms were constant in all the plates from all the 

 galls. Slant agar cultures were made, however, from a selection of 

 such colonies as appeared, and from these subcultures inoculations 

 were made by means of needle pricks into both old and young stems 

 and leaves of healthy daisy plants. An occasional gall was found at 

 or near the point of inoculation, but they were so few and so uncertain 

 as to their formation that the inoculations were considered as having 

 no significance. 



The possibility of these growths being due to bacteria was therefore 

 temporaril}" abandoned (Dr. Townsend) and various attempts 

 were made to produce them by mechanical injuries practiced upon 

 both young and old plants. Notches varying in number and extent 

 were cut with a sharp knife into the sides of main stem and branches. 

 The mam stem was cut off at different distances from the ground. 

 In some instances the entire top was removed, and in other instances 

 only the top of the main stem was cut off. Other injuries of this 

 nature graded between these two extremes. Branches were also cut 

 off at different distances from the main stem, and some were simply 

 clipped without cutting off. Parts of leaves were cut off. The main 

 stems were injured near the base by jabbing with the point of a knife. 

 Some stems and branches were broken off, while others were simply 

 broken and left hanging by a portion of the tissue. In addition to the 

 injuries mentioned, combinations of these were made upon healthy 

 plants in various degrees of severity until we had 20 series of 

 simple and compound injuries. These were all started in the patho- 

 logical greenhouse upon plants produced from cuttings from healthy 

 marguerites. Abnormal growths appeared on some of the injured 

 plants, but they were not produced with any degree of regularity or 

 certainty, and the growths rarely occurred exactly at the point of 

 injury. Furthermore, the abnormal formation when occurring at 

 the point of injury had more the appearance of callous growths than 

 of the original daisy galls. 



About this time (May, 1906) one of us observed, in studying micro- 

 tome sections stained with anilin compounds having a strong affinity 

 for bacteria, that while no distinct bacteria could be made out, never- 

 theless that part of the section lying deepest, i. e., bordering on the 

 sound tissues, took the stain much heavier than the rest of the gall, 



213 



