The most important constituents of the bile, viz., the bile acids and pigment, may be detected by appropriate tests, which are in common practical use: -

1. Pettenkofer's Test For The Bile Acids

To a fluid containing either or both bile acids, or any solution of cholic acid, add some cane sugar, and then slowly, drop by drop, strong sulphuric acid. The solution turns to a cherry-red and then changes to purple. As other substances, such as albuminous bodies, give under this treatment a similar color, in order to make the reaction a trustworthy test for bile salts, the two characteristic absorption bands given by the spectroscope should also be observed.

The following is said to be a characteristic method: Rinse out a porcelain capsule successively with the fluid to be tested with weak sulphuric acid, and with a weak solution of sugar, then heat to 700 C, when the capsule turns purple.

2. Gmelin's Test For The Bile Pigments

Gmelin's Test For The Bile Pigments depends upon the fact that during the stages of oxidation the bilirubin undergoes a series of changes in color which follow the sequence of the familiar solar spectrum. Place a few drops of the fluid to be tested on a white surface (capsule or plate), and allow a drop of nitric acid, yellow with nitrous acid fumes, which make it more oxidizing, to run into it; as they mingle together the rainbow-like play of color appears. This, when watched, will be found to consist of a series of changes to green, blue, violet, red and yellow.

This can also be observed by allowing the acid to trickle gently down the side of a test tube fixed in an inclined position so that it cannot be shaken: the play of color can then be seen starting from the point of junction of the two fluids.