148 
EIGHTH REPORT. 
With the hope of throwing some light upon this question from the 
experimental side, a series of investigations have been undertaken and 
are now in progress in our laboratory aimed primarily at the antag¬ 
onism, if such it be, existant between different soil bacteria and the 
germ producing the nodules of the alfalfa plant. One of these experi¬ 
ments only will be reported upon here and that of a more or less pre¬ 
liminary nature. 
The organisms selected for this first work were B. ramosus and Ps. 
radicicola, (alfalfa). A twenty-four hour bouillon culture of each germ 
was prepared and a known number of cells from each culture were in¬ 
troduced into one of two flasks of sterile salt solution. We then had 
a suspension of alfalfa germs containing a known number of cells in 
one flask and a similar suspension of B. ramosus in the other. One 
c. c. of the alfalfa suspension with its known bacterial content was then 
used to inoculate 99 c.c. of a nutrient sugar solution contained in a 
200 c. c. Erlenmeyer flask; a second flask of sugar solution as above 
was inoculated with one c. c. of the B. ramosus suspension; into a third 
flask of the same medium was introduced one c. c. each of the alfalfa 
and B. ramosus suspensions. We had now one flask with the alfalfa 
alone, one with ramosus alone and the third containing both organ¬ 
isms. These were kept at a temperature of 28° C. for twenty-four hours, 
at the end of which time, quantitative dilution plates were made in a 
sugar agar and the relative. counts determined later. Again after 48 
hrs, 72 lirs., 96 hrs., five days and 6 days, dilution plates were made from 
The three flasks described above and their counts determined as given 
in the table below. Four different lots of cultures, prepared precisely 
ns here described, have been worked with and the counts made for 
periods as previously given, the results of which will follow. 
During all of our investigations the cultures in flasks have been 
kept as near 28° C. as possible, that being the optimum for alfalfa. 
Some difficulty was experienced at first with the B. ramosus spreading 
on the plates when kept at this high temperature and so in all sub¬ 
sequent work the plates have been allowed to develop at approximately 
23°C. 
This temperature has another advantage aside from eliminating 
spreaders for at 23° C. the B. ramosus colonies are plainly visible after 
48 hrs., and can be counted, while the alfalfa colonies seldom appear 
before the fourth day. By this means it was a very simple matter to 
check the counts on the plate said to contain both organisms. Aside 
from this temperature relation, the colonies of each germ are so very 
characteristic that no difficulty was ever experienced in deciding which 
was which. 
