234 LEVINE: STUDIES ON PLANT CANCERS—IV 
medium was then inoculated by pricking the surface of the agar 
in five to eight different places with a needle containing a sub- 
culture of Bacterium tumefaciens. After the bacteria had mul- 
tiplied for from two to forty days the surface of the agar was 
gently scraped to avoid as far as possible the removal of portions 
of the surface of the agar. I employed two methods for inocu- 
lating my plants. The first, a modified form of the commonly 
used pricking method, and the second, the trocar method. In 
the first method the cylinder of a medium-sized hypodermic 
needle was filled with paraffin so as to convert the point into a 
spoon-like container. This was dipped (into an emulsion of 
B. tumefaciens), so that the spoon end of the needle alone 
contained the bacteria. This was then gently stuck into the 
plant at a desired point, five to ten times, until all the emulsion 
was removed into the plant. For larger measured quantities of 
bacteria the second method was used. A plug of tissue 3-5 mm. 
long and 0.5 mm. in diameter was removed from the part of the 
plant where the inoculation was to be made by a trocar. The 
suspension of the crown gall organism was then introduced 
through a graduated hypodermic syringe. Numerous controls 
were made by removing a plug of tissue and filling the cavity 
with sterile water. 
All mother emulsions consisted of 4 cc. of such culture scrap- 
ings of B. tumefaciens, to which was added 2 cc. of sterile water 
to facilitate the removal of the bacteria from the surface of the 
agar. The following dilutions of these cultures were made and 
will be referred to in the text by number. 
ulsion Ia is from a culture of Bacterium tumefaciens 21 days old. 
Subdilutions of this culture were made by the addition of sterile water in the 
following proportions: 20, 45 35 T2210. I, 3 3 20. 
_ Emulsion Ifa is from another set of cultures of Bacterium re aa 
21 days old. JJb consisted of a part of the emulsion, J/a, dilut 
equal part of sterile water. In J/c, the dilution was made in the iid t3. 
Emulsion IIIa is from a third group of cultures grown at another later 
period used at the age of 21 days. In IIIb, the ig is made in the pro- 
portion 1 : 5;in J/Jc, 1 : 10;in IJJd, 1 : 15; in II Te, 1 
Emulsion IV represents still another set of ae 21 days old. This 
was used undiluted. 
Emulsion V is from cultures of Bacterium tumefaciens 40 days old. 
Emulsion VIa is an e:nulsion from young ~_— 2 days old. VJb was 
diluted 2-25; Vie, 1:10; Vid, 1:20; Vie, 1:4 
Emulsion VII is an undiluted mother seit seven days old. 
