Nov. 15,1925 Value of Indicators in Studying Legume Nodule Organisms 999 
According to change in reaction, the cultures fall into two groups: 
Acid production with strong reduction, and alkaline production with 
little or no reduction. 
The cultures from alfalfa and sweet-clover nodules were the only 
ones that showed an acid reaction. Within the first few days after 
inoculation the organisms produced an alkaline reaction in milk, 
but later they began to reduce the litmus, and still later the milk 
began to turn pink. The rate of acid production was much faster 
with freshly isolated strains. The tubes of milk usually remained 
reduced at the bottom, while the upper layer was pink. Some 
cultures kept the litmus reduced completely below the serum zone. 
The cultures of the second group—which included pea, vetch, 
clover, bean, lupine, cowpea, lima-bean and soy-bean organisms— 
produced alkaline reactions (Table I). The milk cultures showed 
a change in reaction within a few days after inoculation. The most 
marked changes were produced by soy-bean, lima-bean, cowpea, and 
pea cultures. Some clover and pea strains reduced the litmus com¬ 
pletely after about two weeks. These were cultures which had been 
cultivated in the laboratory for a number of years. Other clover, 
pea, and bean strains reduced the litmus slightly, usually at the 
bottom of the tube. Cultures of cowpea, lima bean, and soy bean 
did not reduce the litmus. A film growth over the surface was formed 
by some cultures of clover, pea, and vetch. This film formation was 
especially noticeable with strains which showed a marked reduction 
of the litmus. 
The characteristic reaction of Bacillus radiobacter is shown quite 
as well in litmus milk as in milk without the indicator. 
The effect of the growth of these cultures on the hydrogen-ion con¬ 
centration of the milk after 47 days was studied. The determina¬ 
tions reported below were made by the electrometric method: 
Ph 
Uninoculated;_ 6. 55 
Alfalfa, 108_ 5. 18 
Do. 109_-_ 5.31 
Soy bean, 164__ 7. 75 
Do. 168_ 8.37 
Lupine, 150--- 7. 55 
Lima bean, 142__ 7. 85 
Cowpea, 143_ 8. 08 
The alkaline reaction produced by the growth of soy-bean, cow¬ 
pea, and lima-bean nodule organisms in milk probably accounts for 
the liquefying effect on the milk casein noted by Lohnis and Hansen. 3 
These investigators noted a slow digestion of the casein by these 
organisms after a long time. Preliminary experiments by the 
writer indicate that the nodule organisms have very little, ii any, 
effect on the milk casein. However, the alkaline reactions pro¬ 
duced by soy-bean, cowpea, and lima-bean nodule organisms, as 
shown above, are sufficient to cause a slow dissolving of the milk 
casein, with a resulting decrease in opacity of the milk. 
A serum zone was formed by the alfalfa, sweet-clover, clover, pea, 
vetch, and bean cultures. Clover, pea, and vetch cultures would 
often produce a serum zone in two or three days, while some strains 
of alfalfa, sweet-clover and bean organisms would often require 
3 Op. cit. 
