236 PHYSIOLOGICAL CHEMISTRY 



shallow, flat-bottomed dish and allow to stand 6-24 hours. The presence of 

 hydrobilirubin will be indicated by a deep red color being imparted to the par- 

 ticles of feces containing this pigment. This red color is due to the formation 

 of hydrobilirubin-mercury. If unaltered bilirubin is present in any portion of 

 the feces that portion will be green in color due to the oxidation of bilirubin to 

 biliverdin. 



-Another method for the detection of hydrobilirubin is the following: Treat 

 the dry feces with absolute alcohol acidified with sulphuric acid and shake 

 thoroughly. The acidified alcohol extracts the pigment and assumes a reddish 

 color. Examine a little of this fluid spectroscopically and note the typical 

 spectrum of hydrobilirubin (Absorption Spectra, Plate II). 



8. Bilirubin. 1 (a) Gmelin's Test. Place a few drops of concentrated 

 nitric acid in an evaporating dish or on a porcelain test-tablet and allow a few 

 drops of the feces and water to mix with it. The usual play of colors of Gmelin's 

 test is produced, i.e., green, blue, violet, red, and yellow. If so desired, this 

 test may be executed on a slide and observed under a microscope. 



(b) Huppert's Test. Treat the feces with water to form a semi-fluid mass, add 

 an equal amount of milk of lime, shake thoroughly, and filter. Wash the precipi- 

 tate with water, then transfer both the paper and the precipitate to a small beaker 

 or flask, add a small amount of 95 per cent alcohol acidified slightly with sulphuric 

 acid, and heat to boiling on a water-bath. The presence of bilirubin is indicated 

 by the alcohol assuming a green color. 



Steensma advises the addition of a drop of a 0.5 per cent solution of sodium 

 nitrite to the acid-alcohol mixture before warming on the water-bath. Try this 

 modification also. 



9. Bile Acids. Extract a small amount of feces with alcohol and filter. 

 Evaporate the filtrate on a water-bath to drive off the alcohol and dissolve the 

 residue in water made slightly alkaline with potassium hydroxide. Upon this 

 aqueous solution try any of the tests for bile acids given on page 208. 



10. Casein. Extract the fresh feces first with a dilute solution of sodium 

 chloride, and later with water acidified with dilute acetic acid, to remove soluble 

 proteins. Now extract the feces with 0.5 per cent sodium carbonate and filter. 

 Add dilute acetic acid to the filtrate to precipitate the casein, being careful 

 not to add an excess of the reagent as the casein would dissolve. Filter off the 

 casein and test it according to directions given on page 321. Casein is found 

 principally in the feces of children who have been fed a milk diet. Mucin would 

 also be extracted by the dilute alkali, if present in the feces. What test could 

 you make on the newly precipitated body to differentiate between mucin and 

 casein? 



11. Nucleoprotein. Mix the stool thoroughly with water, transfer to a flask, 

 and add an equal amount of saturated lime water. Shake frequently for a few 

 hours, filter, and precipitate the nucleoprotein with acetic acid. Filter off this 

 precipitate and test it as follows: 



(a) Phosphorus. Test for phosphorus by fusion (see page 129). 



(b) Solubility. Try the solubility in the ordinary solvents. 



(c) Protein Color Test. Try any of the protein color tests. 



1 The detection of bilirubin in the feces is comparatively simple provided it is not ac- 

 companied by other pigments. When other pigments are present, however, it is difficult to 

 detect the bilirubin and, at times, may be found impossible. 



