212 



PHYSIOLOGICAL CHEMISTRY 



(e) Neukomm's Modification of Pettenkofer's Test. To a few drops of 

 diluted bile in an evaporating dish add a trace of a dilute sucrose solution and one 

 or more drops of dilute sulphuric acid. Evaporate on a water-bath and note the 

 development of a violet tolor at the edge of the evaporating mixture. Discontinue 

 the evaporation as soon as the color is observed. 



(f) Peptone Test (Oliver). To 5 c.c. of diluted bile add 2-3 drops of acetic 

 acid, filtering if necessary. Add an equal volume of a i per cent solution of 

 Witte's peptone to the acid solution. A precipitate is produced which is insoluble 

 in excess of acetic acid. This precipitate is a compound of protein and bile acids. 



6. Crystallization of Bile Salts. To 25 c.c. of undiluted bile in an evaporating 

 dish add enough animal charcoal to form a paste and evaporate to dryness on a 

 water-bath. Remove the residue, grind it in a mortar, and transfer it to a small 

 flask. Add about 50 c.c. of 95 per cent alcohol and boil on a water-bath for 20 

 minutes. Filter, and add ether to the filtrate until there is a slight permanent 

 fcloudiness. Cover the vessel and stand it away until crystallization is complete. 

 Examine the crystals under the microscope and compare them with those shown in 

 Fig. 61, page 208. Try one of the tests for bile acids upon some of the crystals. 

 f- 7. Analysis of Biliary Calculi. Grind the calculus in a mortar with 10 c.c. 

 of ether. Filter. 



Filtrate I. 



Add an equal volume of 95 per cent alco- 

 hol 1 to the ether extract, allow the mix- 

 ture to evaporate and examine for choles- 

 terol crystals (Fig. 63, page 213). (For 

 further tests see Experiment 8, below.) 



I 



Residue 1. 



(On paper and in mortar.) 



Treat with dilute hydrochloric acid and 

 filter. 



Filtrate II. 



Test for calcium, phosphates, and iron. 

 Evaporate remainder of filtrate to dry- 

 ness in porcelain crucible and ignite. Dis- 

 solve residue hi dilute hydrochloric acid 

 and make alkaline with ammonium hy- 

 droxide. Blue color indicates copper. 



Residue II. 



(On paper and in mortar.) 

 Wash with a little water. Dry the filter 

 paper. 



Treat with 5 c.c. chloroform and filter. 



Filtrate III. Residue in. 



Bilirubin. (On paper and in mortar.) 

 (Apply test for 

 bile pigments.) 



Treat with 5 c.c. of hot 

 alcohol. 



' ^ '. ~ ' . . . Biliverdin, 



8. Tests, for Cholesterol. 



(a) Microscopical Examination. Examine the crystals under the microscope 

 and compare them with those shown in Fig. 63, below. 



(b) Sulphuric Acid Test (SalkowsM). Dissolve a few crystals of cholesterol 

 in a little chloroform and add an equal volume of concentrated sulphuric acid. 

 A play of colors from bluish-red to cherry-red and purple is noted in the chloro- 

 form while the acid assumes a marked green fluorescence. 



1 The alcohol is added because of the fact that it is often found that crystallization from 

 pure ether does not yield typical cholesterol crystals. 



