26 ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 
After the litmus has been reduced it regains its color upon standing due to oxida- 
tion by the oxygen of the air. 
Brom Cresol Purple Milk 
Brom crésol purple is the laboratory name of Di-bromo-ortho-cresol-sulfon- 
phtalein. As a substitute for litmus in milk media, brom cresol purple has the 
advantage of having a known chemical composition and is obtainable in the pure 
state. In the concentration used, it has little, if any, inhibitory effect upon the 
growth of bacteria. It is a more sensitive indicator for the reaction changes pro- 
duced in milk by bacteria than is litmus. Brom cresol purple is not readily re- 
duced by bacteria or by heat. This is an added advantage for most purposes, but 
from the standpoint of detecting the reducing power of bacteria it is a disadvan- 
tage, since brom cresol purple is not reduced as is litmus by bacterial action. 
Brom Cresol Purple Solution 
A stock solution for use in brom cresol purple milk is made as follows: 
Grind in a glass mortar 0.5 gram of brom cresol purple; add to the fine 
powder 14 cc. of a N/10 NaOH solution and stir well. Transfer to a flask. 
Wash the mortar with 75 c.c. of distilled water and pour the wash water into 
the same flask. Shake until solution is complete. Make up to 100 c.c. with dis- 
tilled water. 
Ten c.c. of this solution added to a liter of milk gives a concentration of 
0.005 per cent. 
EXeErcIsE 41 
REDUCTION OF NITRATES 
Many bacteria have the power of reducing nitrates to nitrites; a few are 
able to break down nitrates to gaseous nitrogen, either free nitrogen or oxides of 
nitrogen, 
Inoculate Smith fermentation tubes containing nitrate broth with Strepto- 
coccus lactis, Proteus vulgaris, and Pseudomonas pyocyaneus. The composition 
of the nitrate broth is: 
0.3% beef extract 
1.0% peptone 
1.0% KNO, 
Distilled water 
Incubate two days. If gas is produced in any of the tubes, test as in Exer- 
cise 37. Test all of the cultures for nitrites, using Trommsdorf’s reagent. 
Test for Nitrites 
Place 3 drops of Trommsdorf’s solution in a depression on a spot plate. Add 
1 or 2 drops of sulphuric acid (1:3). Remove a loopful of the solution to be 
