METHODS FOR MICROSCOPIC STUDY OF BACTERIA 251 
Test.— Add 5 cc. solution of No. 1 to 10 cc. culture, then add o cc. 
of solution Xo. 2, and shake well. A red color develops within five 
minutes if the test is positive. Gore' has modified the test as follows: 
Remove the plug from the culture tube (white, absorbent cotton must 
be used), moisten the plug with 4 to 5 drops of solution Xo. 2 and with a 
like amount of Xo, 1. Replace the plug, and stand the tube upright 
in a water-bath. Heat for fifteen minutes. Do not allow the culture 
to touch the cotton plug. A red color developing on the cotton 
indicates indol. 
Sometimes a blue color, or a purplish color, develops if solution Xo. 1 
only is used. The blue color is probably due to scatol and the pur- 
plish color to a mixture of scatol and indol.^ 
Gnezda Oxalic Acid Test. — JYip strips of filter paper in a warm, sat- 
urated solution of oxalic acid. Dry. Insert a strip in the culture tube 
against the side, at the mouth, and replace cotton plug after inoculation. 
Indol, which is volatile, causes the oxalic acid crystals on the paper to 
turn pink. X on-volatile indol acetic acid, which also gives a reddish 
color, is eliminated by this procedure. 
(b) Carbohydrate Broths.— The addition of sugars, as glucose, lac- 
tose, saccharose; or of alcohols, as glycerol or mannitol, to plain broth 
media, greatly enriches the medium in non-nitrogenous substances 
which may be readily utilizable sources of energy for bacteria. It is 
hardly necessary to emphasize the importance of purity in all sugars and 
other carbohydrates intended for bacterial purposes, nor the fallacy 
of attempting to determine the action of bacteria upon specific car- 
bohydrates in media not freed from muscle sugar (glucose). The 
use of serum as a basis for fermentation media frequently introduces 
a source of error, because blood serum normally contains about 0.08 
per cent of glucose, an amount quite sufficient to give rise to con- 
siderable amounts of acid.'^ 
The observations made in carbohydrate media are usually restricted 
to: 
(fl) Change in reaction. 
(6) Gas formation in fermentation tubes, and growth in the closed 
arm as well. 
I Indian Jour. Med. Res., 1921, 8, 505. 
' Herter and Foster: Jour. Biol. Chem., 1906, 1, 251. A good discussion of indol 
methods will be found in the Jour. Bacteriol., 1925, 10, 459, by Kulp. 
^ The significance of fermentation media for the classification and identification of 
bacteria depends upon their content both of protein and carbohydrate. Bacteria derive 
their energy requirements from carbohydrate, if it is utilizable, but of course they must 
obtain their "Bausteine" from the nitrogenous constituents. If the carbohydrate 
cannot be utilized, both structural and energy requirements are derived from the pro- 
tein constituents. Bacteria vary greatly in their ability to ferment carbohydrates; 
some tj-pes, as Bacillus alcaligenes, do not appear to ferment even glucose. Bacillus 
lactis aerogenes, on the contrary, can ferment hexoses, bioses and even starches. The 
fermentability of a carbohydrate depends apparently upon its stereo-isomeric con- 
figuration, and relatively slight differences in the configuration of similar carbohydrates 
may determine their value for specific organisms as sources of energy. This point is 
discussed somewhat later in this section. 
