THE VALUE OF LITMUS, BROM-CRESOL PURPLE, AND 
JANUS-GREEN MILK IN A STUDY OF THE NODULE 
ORGANISMS OF LEGUMINOSAE 1 
By J. W. Stevens 
Assistant in Agricultural Bacteriology , University of Wisconsin 
INTRODUCTION 
The value of milk in the study of the nodule bacteria of legumes 
has been shown by Lohnis and Hansen. 2 They found that in milk, 
organisms with peritrichous flagella from alfalfa, sweet clover, clover, 
pea, vetch, lupine, and bean formed a cleat serum zone on top 2rttfv5 
mm. thick, while the milk below remained nearly unchanged; and 
that organisms of the second group, such as cowpea, peanut, lima 
bean, and soy bean, which have only one flagellum, formed no serum 
zone but that later there was a slow digestion of the milk. They also 
f minted out the value of milk in distinguishing Bacillus radiobacter 
rom nodule bacteria of legumes. 
The interesting reaction of the nodule bacteria in milk suggested 
the possibility of improving plain milk by the addition of some 
indicator, which would show reduction and change in reaction. In 
an attempt to improve the medium, litmus, brom-cresol purple, and 
Janus-green milk media were prepared and their value compared 
with jflain milk. 
Only fresh skim milk was used as culture medium. This was 
sterilized in an Arnold steamer at 100° C. for 30 minutes on four 
successive days. Aqueous solutions of the indicators were sterilized 
separately and added to the milk. For a comparison of plain and 
litmus milk four parallel tubes of each were inoculated with the same 
culture, while with brom-cresol purple and J anus green only two tubes 
of each culture were used. Pure cultures of nodule bacteria from 
pea, vetch, clover, garden bean, lima bean, cowpea, soy bean, alfalfa, 
and sweet clover were used. All cultures were incubated at 28° C. 
Of the three indicators, litmus proved by far the most valuable, 
Brom-cresol purple and Janus green failed to bring out any differences 
hot shown by litmus. Brom-cresol purple showed changes in reac¬ 
tion, while Janus green showed a reduction. As Janus green remains 
reduced permanently it is of less diagnostic value than litmus» 
Brom-cresol purple was not reduced by any of the nodule organisms. 
This indicator retarded the growth of the organisms, in some cases 
completely. High concentrations of litmus in milk had no noticeable 
inhibitive effect on the growth of these organisms, and they showed 
changes in reaction equally as well as brom-cresol purple, and in 
indicating reduction they were as satisfactory as Janus green*. 
After one to three weeks most of the cultures showed an alkaline 
reaction in brom-cresol-purple milk. However, after five weeks some 
1 Received for publication May 6, 1925; issued January, 1926. Published with the approval of the 
Director of the Wisconsin Agricultural Experiment Station. . ^ _ 
* Lohnis, F., and Hansen, R. nodule bacteria of leguminous plants. Jour. Agr. Research 20: 543- 
556, illus. 1921. 
Journal of Agricultural Research, 
Washington, D. C. 
Vol. XXXI, No. 10 
Nov. 15, 1925 
Key No. Wis.-31 
74780- 26f-7 
( 997 ) 
