24 CKOWN-GALL OF PLANTS. 



as though the Hving bacteria might be lodged most abundantly in 

 this portion. It was suggested, therefore, that for the next series of 

 plates deeper tissues should be used. Six sound daisy galls varying 

 in size from 2 to 20 mm. in diameter were therefore selected by Miss 

 Haskins and carefully washed in distilled water with a firm brush, 

 rinsed in twice distilled water, put into mercuric chlorid water 

 (1 : 1,000) for 45 seconds, rinsed in sterile water and each knot then 

 placed in a test tube containing 10 c. c. of sterile bouillon. In cutting 

 these galls, a small portion of the stem at the point of attachment 

 of the gall was also removed with the gaU. After placing these galls 

 in bouillon they were cut and mashed as much as possible with a ster- 

 ile knife and a sterile glass rod. Some of the more woody portions 

 it was impossible to crush thoroughly. However, from these six 

 bouillons, each inoculated from a separate gall, 19 agar-plate cultures 

 were made, three from each tube except No. 1. In 48 hours all the 

 bouillon tubes were clouded and a yellow organism developed during 

 the same period in four of the six groups of plates; that is, four of the 

 knots had produced agar-plate colonies in 48 hours, the plates being 

 kept at room temperatures of from 20° to 25° C. On the fifth day 

 after the plates were poured a few small, round, white colonies ap- 

 peared in each plate in five of the six series. Slant agar and potato 

 cylinder cultures were made from both the yellow and white colonies, 

 also cultures in litmus milk. The indications were that three kinds 

 of yellow colonies had formed, and that all the white ones were alike. 

 On June 1 inoculations were made from each of the 4 organ- 

 isms into the stems of young healthy daisy plants growing in the 

 pathological greenhouse. The inoculations were made at the top, 

 middle, and base of the stem in each case. For this purpose young 

 slant agar subcultures were used. The portion of the stem to be 

 inoculated was washed with corrosive sublimate w^ater (1 : 1,000) and 

 then with sterile water. The growing organism was smeared upon 

 the stem with a sterile platinum needle and pricked into the tissues 

 by means of a sterile steel sewing needle. Control pricks were made 

 with a sterile needle on other daisy plants for comparison. 



On June 8 another set of healthy plants, older than the former set, 

 was inoculated with fresh cultures of the four organisms in the 

 manner described above. 



On June 18, in the first series (those of June 1) a distinct elevation 

 (knotty growth) was visible at each point where an inoculation had 

 been made with the white organism, but no change had taken place 

 in any of the plants inoculated with the yellow organisms nor in any 

 of the control plants. 



In the second series on June 23, that is, 15 days after inoculation, 

 slight elevations were visible at all points where the white organism 



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