CAUSE AND NATURE OF CROWN-GALL 41 



mass of soft, abnormal tissue generally known under the name 

 of crown-gall. 



For the purpose of successful study of a plasmodium in its 

 earliest stages in the host, material must be carefully selected. 

 In my investigations I found young galls from i 1 ? to 2 milli- 

 meters in diameter the most desirable. In order to obtain speci- 

 mens free from soil and saprophytes, I grew the galls by inoculat- 

 ing almond seedlings and placing them in water cultures for a 

 period of three or four weeks. In growing galls on inoculated 

 seedlings in water culture, the point of infection should be from 

 one and a half to two inches above the water line in the jar. 

 They should be kept in a cool room and the water changed 

 every two or three days. Material obtained in this way is par- 

 ticularly desirable for fixing, imbedding, and cutting into serial 

 sections. Both free-hand and serial sections of the young galls 

 were examined in large numbers. Free-hand sections were 

 studied in water and in a weak solution of glucose. The serial 

 sections were from material imbedded in paraffine, and were 

 stained and mounted ill balsam in the usual way. Various 

 fixing fluids were tried, including corrosive sublimate, chromic 

 acid, and Flemming's fluid of various strengths. After repeated 

 experiments, Flemming was found to be the most desirable, es- 

 pecially the strong solution. The sections were stained on the 

 slide, the triple stain of Safranin, Gentian violet, and Orange 

 was the most satisfactory. The tissue of the young gall is so 

 thin- walled that.it is necessary to carry the sections from the 

 Gentian violet to and through the Orange with great rapidity 

 in order to obtain the best results. 



Thin free-hand sections of the young gall examined in water 

 show the characteristic thin-walled parenchyma with scattered 

 centers of meristematic tissue as previously described. At this 

 stage of the gall development there is no starch in any of its 

 tissues. Many abnormally large cells are filled with compound 

 crystals, probably of calcium oxalate. These crystals are much 

 more frequent than in the normal tissue of the root. The cells 

 as a rule have a surprisingly small amount of organized cell 

 contents when compared with the size of the cell lumen, and 

 the nuclei are clearly discernible in fresh unstained specimens. 



4 



