24 The Colorado Experiment Station 
Litmus Milk .—Prepared by adding to plain milk one per cent 
of a solution of azolitmin made by dissolving i gram of azolitmin 
in 40 c.c. of distilled water and kept at 37.5 0 C. for 12 to 18 hours. 
GREENHOUSE EXPERIMENTS. 
The constant occurrence of characteristic white colonies, in 
such a large percentage of our plates, was sufficient to make us 
suspicious that the micro-organisms making up such colonies were 
the immediate cause of the disease. However, the crucial test of 
a pathogenic organism is its power to reproduce the given disease 
when introduced in pure culture into its normal host. Accordingly, 
we have fulfilled this requirement by making a large number of in¬ 
oculations upon alfalfa plants under greenhouse conditions, and by 
this means we have been able to establish Ps. medicaginis, n. sp. 
beyond the remotest shade of possible doubt, as the unquestionable 
cause of the trouble. We have reproduced the infection in from 
five-to seven days with practically its characteristic field symptoms, 
and we have been able to follow its progress through the different 
changes up to the blackening and complete destruction of the stem 
after six weeks. 
In the plant inoculations, which are described below, we have 
employed three different germs. These are the only ones which 
we have met with in our isolations, and for convenience they are 
here referred to as “Yellow Colony,” “Orange Colony” and “White 
Colony,” the last, Ps. medicaginis, n. sp., having been shown to be 
alone responsible for the disease. 
Successful inoculations have been obtained by scarifying the 
epidermis of the stems with a sterile scalpel, and then immediately 
smearing the freshly exposed, moist surface with a 48 to 72 hour 
agar culture of the organism. Another method has been to prick 
the stem at intervals of about one centimeter with a needle pre¬ 
viously dipped into the growth of a 48 to 72 hour agar streak. 
Again, we have rubbed the culture over the surface without any 
previous injury to the epidermis. 
No precautions have been taken against the drying out of the 
inoculated areas other than to spray the plants once just after 
inoculation, and once a day thereafter for the next three days 
with sterile water. A water bottle with a fine rose nozzle has been 
used for this w r ork. The spray would collect in large drops, and 
ultimately run down the stems, and in this way moisten the in¬ 
oculated regions. This treatment together with the moist atmos¬ 
phere of the greenhouse seemed adequate. 
Plant Inoculations of June 3, 1909. 
Cultures used were isolated June 1, 1909. 
Pot No. 1.— The epidermis of each of four stems was scarified for a 
space of 10 cm. and smeared with a 48 hour agar culture of Ps. medi- 
