Jan. 29, 1917 
Mechanism of Tumor Growth in Crowngall 
169 
neoplasms (until secondary infections occur), because the tumor cells are 
not visibly injured except that they stain somewhat differently from 
normal embryonic tissues, are unduly excited and, along with greatly 
increased vegetative tendencies, have lost the whole or a great part of 
their polarity (orderly arrangement of cells) and also most of their 
power to differentiate tissues. The most conspicuous results of the 
growth of Bact. tumefaciens in culture media containing grape sugar and 
Witte's peptone are ammonia and alcohol (Alsberg, Brewster, Wood¬ 
ward) and an acid, said by Dr. Carl L. Alsberg, Chief of the Bureau of 
Chemistry, to be acetic acid in case of the Daisy strain with traces of oil or 
of a fatty acid, and by Dr. J. F. Brewster, also of the Bureau of Chemis¬ 
try, one of the fatty acids (but not acetic acid) in case of the hop strain. 
These substances are produced by the bacterium in flasks of Jena glass 
containing only calcium carbonate, water, grape sugar, and peptone, and 
undoubtedly they are also produced by it within the cells of the tumor. 
According to Dr. H. E. Woodward, of the Bureau of Chemistry, small quan¬ 
tities of amins (but not trimethylamin) are produced by the hop strain— 
that is, primary or secondary amins. 1 Probably the organism also 
1 Since these two sets of analyses were made Dr. Alsberg has kindly had further analyses made for me of 
Bad, tumefaciens plated from Flats poplar, from Massachusetts rose, and from another Paris daisy gall. 
These were flask cultures grown over calcium carbonate in distilled water containing 1 per cent Witte’s pep- 
tonum siccum, and 1 per cent Merck’s c. p. dextrose, sterilized by discontinuous steam heat on 4 consecutive 
days and allowed to stand some days before inoculation. These analyses, which were made by Drs. Brewster 
and Woodward, at the end of 8 to 9 weeks, show, as in the case of the previous analyses of the hop strain, that 
alcohol, ammonia, and amins are formed and small quantities of fatty adds. Formic add was detected 
in each of the nine flasks. No acetic add was found in flasks inoculated with the daisy strain, but it 
occurred in those inoculated from poplar and rose. Aldehyde and acetone were also detected in each 
flask. No trimethylamin was found. The analyses of a second series of the hop strain were made in 
January on cultures somewhat older than the first series. (See page 186.) 
In each instance I personally inoculated the flasks with great care, using agar subcultures made originally 
from single poured-plate colonies, flaming the necks of the flasks thoroughly in advance, turning them 
down on their sides to inoculate and keeping them open for a moment only in still air in a clean culture 
chamber, but even so there are always slight chances of contamination. To further eliminate these chances 
several flasks were used for each strain, several were held uninoculated for control, and the others were 
watched to see that growth came on normally for this organism. Finally agar poured-plates were made 
from each inoculated flask to judge of its continued purity and subcultures from each were tested out on 
plants and found to be infectious. The flasks, including those which were not inoculated and all of which 
had remained sterile, were then turned over to the chemists, who analyzed each one separately, with con¬ 
cordant results except as indicated below. 
CrfEMISTS’ REPORT OF ANALYSES OF FLASK CULTURES 
“Three flasks each of cultures marked Flats Poplar, Rose, and Resistant Daisy, dated October 21, 1916, 
were received from Dr. Smith on December 16, 1916. With these were also three control flasks which 
had not been inoculated. 
“ The flasks were opened and each worked through in order until finished, beginning December 18, the 
methods being as follows: 
“The contents were filtered off from undissolved calcium carbonate. A small portion of the filtrate 
acidified with acetic acid and treated with a few drops of ammonium oxalate solution gave an abundant 
precipitate of Ca-oxalate showing that much calcium carbonate "had been dissolved. This is true of all 
the flasks which had been inoculated. Those not inoculated gave no test for dissolved Ca. The remainder 
of the cloudy filtrate was distilled over milk of lime. Some of the distillations were done with the free 
flame, others at diminished pressure to determine whether the same products could be found at the lower 
distilling temperature (4o°-so° C.). In either case the products were the same. 
“Small portions of the alkaline distillate were tested for alcohol, acetone, and aldehyd, and the main 
portion, after neutralizing, was given to Dr. Woodward to test for volatile alkali. 
“ Acetone was first indicated by the formation of iodoform at the ordinary temperature when 2 drops of 
10 per cent potassium hydroxid solution and sufficient iodine solution to produce a faint yellow color 
