FEBRUARY 2, 1912] 
often only by using a hundred, a thousand, 
or a hundred thousand times too much ma- 
terial, if one were working with other organ- 
isms. The second obstacle is the fact that 
the living bacteria in the tumor tissue occur 
for the most part in a paralyzed condition, 
either as involution forms or in some other 
form which does not grow readily when 
plates are made. Cultures were made 
every few weeks from crown-gall tissue for 
two years and numerous and various bac- 
teria were obtained on these plates, pricked 
off for sub-culture, studied microscopically 
and culturally, and inoculated into the 
plant with negative results, these organ- 
isms being the saprophytes which usually 
accompany crown-gall. The plates were 
usually discarded after three or four days, 
and so the work went on. If, however, one 
inoculates copiously as described, and 
waits a week or ten days for the paralyzed 
organisms to recover their vigor, he will 
then obtain colonies of the parasite. 
Two questions arise: (1) Why does an 
organism which produces such striking re- 
sults occur in the tissue in such small num- 
bers? (2) What paralyzes it so that when 
agar plates are made from the tissues the 
colonies do not appear until the fourth, 
fifth, sixth, eighth or tenth day, and some- 
times not until the twentieth day? These 
questions have received a good deal of 
thought. After a time we discovered that 
when the organism is grown in bouillon or 
other media containing sugar an acid is 
produced, and it then occurred to me that 
this acid might be the cause of the death 
of a large proportion of the organisms in 
the cells, and of the paralyzing of the re- 
mainder. Peptone water flask cultures 
of the organism were then grown in the 
presence of sugar and turned over to the 
chemist, who reported that the acid present 
was acetic acid. We found that after a 
time all the organisms in such cultures 
SCIENCE 
169 
were dead, and a microscopic examination 
showed that a large proportion of them oc- 
curred in the form of irregular club- 
shaped or Y-shaped bodies, i. e., they had 
passed over into involution forms preced- 
ing their death. Subsequently we found 
that on adding dilute acetic acid to fresh 
cultures of the organism either on agar or 
in bouillon we could at will produce these 
involution forms. Ordinarily it was 
found on making poured plates from such 
cultures that all the organisms were dead, 
but by further experimenting we learned 
that if we added just the right quantity of 
acid, involution forms were produced and 
a portion of the bacteria killed, but that 
some remained alive and those which re- 
mained alive were paralyzed, coming up 
on the agar plates in the same slow man- 
ner as those from the interior of the 
tumors. I should have stated that al- 
though the organism comes up slowly from 
the crown-gall on agar-poured plates, sub- 
cultures from such colonies grow as read- 
ily as from any other easily cultivable or- 
ganism, B. coli, for example, showing 
clearly that the initial slow growth is not a 
peculiarity due to differences in culture- 
media or inherent in the organism, but 
only one due to its previous environment 
in the plant cell. 
Do the same phenomena occur in the 
plant cell? Recently from crown-gall of . 
daisy grown for the purpose, the chemist 
has isolated for us an acid which he says is 
acetic acid. We have also found in the 
tissues numerous bacterial Y-shaped bod- 
ies, such as oceur in our flasks when acetic 
acid is present. J think, therefore, we may 
assume, tentatively, at least, that an acid 
in small quantities is formed also in the 
cells of the erown-gall as a by-product of 
the bacterial growth, and that after a 
time this acid stops the growth of the 
multiplying bacteria within the cells ex- 
