214 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
weak acids or bases. The ions of the indicators are not colored the 
same as the undissociated molecules. Many indicators are not 
adapted to the measure of reaction in culture media because the 
presence of proteins and protein derivatives, and of salts, interferes 
with the color changes of such indicators. In protein-salt-free solu- 
tions, however, such indicators would be valuable for the determination 
of reaction, 
Sorensen,! and Clark and Lubs,' have published lists of indicators 
which are quite generally useful for the routine determination of reac- 
tion in cultural media. 
The following table contains such a list, showing the pH values 
between w^hich color changes are of sufficient definiteness to be of 
value in bacteriological procedures. It should be remembered that 
the color changes at the extremes of the pH range are less readily 
differentiated than those within these limits. Thus, phenol red has 
a color range of pH 6.8 to 8.4. The color changes are best detected 
within the range of 7 to 8.2. 
Clark and Lubs, Indicators. ^ 
Indicator. Color change. Range pH.^ 
Tetra brom phenol sulphonphthalein (brom 
phenol blue) Yellow blue 3.2-4.4 
Ortho carboxybenzene azo dimethylanilin 
(methyl red) Red-yellow 4.6-5.6 
Dibrom orthocresol sulphonphthalein (brom 
cresol purple) Yellow-brown 5.4-6.6 
purple 
Dibrom thymol sulphonphthalein (brom thy- 
mol blue) Yellow-blue 6.2-7.4 
Phenol sulphonphthalein (phenol red) . Yellow-red 7.0-8.2 
Phenolphthalein Colorless-red 8.3-9.5 
To determine the reaction of media by the method of hydrogen-ion 
concentration, it is necessary to have a series of clear glass tubes of 
uniform diameter, each containing the proper amount of indicator 
and of solution of definite pH value. A series of such tubes ranging 
from pH 5 to pH 9.4, at suitable intervals, will be found satisfactory 
for the usual reactions met with in bacteriology. 
Preparation of Sta7idard Indicator Sohdions.— The indicators 
tabulated above go into solution best in the presence of dilute alkali. 
Clark^ gives the proper amount of ^ XaOH to add to eft'ect solution 
of the indicator in an amount sufficient to make 25 cc. of the final 
solution. 
1 Enzyme Studies, Biochem. Ztschr., 1909, 21, 131, 201; 22, 352. 
2 Jour. Biol. Chem., 1917, 2, 1, 109, 191. 
' Determination of Hydrogen Ions, 1920. 
'' The effective color range given is somewhat less than that stated by Clark (Deter- 
mination of Hydrogen Ions, 1920). Experience has shown that bacterial cultures 
that are quite turbid are not readily evaluated by indicators at the extremes of their 
color range. This is especially true of the less intense colors, as the yellows. Phenol- 
phthalein is substituted in this list for Clark's cresol- and thymol-blue (pn 7.2-8.8 and 
8.0-9.6). 
* Loc. cit. 
