PRACTICAL EXERCISES 487 



(/) Test for Peptone in Urine (Peptonuria}. Place some of the 

 urine in a beaker on a boiling water-bath for thirty minutes, and 

 saturate with ammonium sulphate crystals. Then boil over a small 

 flame or in an air-bath for half an hour. All the proteins, including 

 peptones, are precipitated. But the peptones can still be redissolved 

 by water, the others not. Filter hot. Wash the precipitate on the 

 filter with a boiling saturated solution of ammonium sulphate. Then 

 extract the residue with cold water, filter, and test the filtrate by the 

 biuret test (addition of very dilute cupric sulphate and excess of 

 sodium hydroxide) . A rose colour indicates the presence of peptone 

 (p. 7), but if the reaction is only a faint one, it may be due to 

 urobilin (Stokvis) . True peptone is rarely found in urine. 



(2) Quantitative Estimation of Coaoulable Proteins (Serum-Albumin 

 and Globulin) (a) Gravimetric Method. Heat 50 to 100 c.c. of the 

 urine to boiling, adding a dilute solution (2 per cent.) of acetic acid 

 by drops as long as the precipitate seems to be increased. Filter 

 through a weighed filter. Wash the precipitate on the filter with hot 

 water, then with hot alcohol, and finally with ether. Dry in an air- 

 bath at 110 C., and weigh between watch-glasses of known weight. 



(b) Method of Roberts and Stolnikow (modified by Brandberg). 

 This method is founded on the fact that the time taken for the white 

 ring to appear in Heller's test depends on the proportion of coagulable 

 protein present. It has been found that when i part of albumin is 

 contained in 30,000 parts of an albuminous solution (0*0033 per cent.), 

 the ring appears in two and a half to three minutes. The amount of 

 dilution of the urine which is necessary to delay the formation of the 

 ring for this length of time is what has to be determined. To do 

 this, proceed as follows : Dilute a portion of the urine (say 5 c.c.) ten 

 times that is, add to it nine times its volume of distilled water 

 from a burette. Place some pure nitric acid in a test-tube with a 

 pipette, taking care not to wet the sides of the test-tube with the 

 acid. Now run on to the surface of the nitric acid some of the 

 diluted urine. Hold the test-tube up against the light from a window* 

 Between the test-tube and the window hold obliquely a dull black 

 surface a few inches from the test-tube, moving it up and down a 

 little below the junction of the urine and acid. Note the interval 

 that elapses before formation of the white ring. If it is more than 

 three minutes, the diluted urine contains less than i part in 30,000, 

 and the undiluted urine less than i part in 3,000 (i.e., less than 0*033 

 per cent.) of coagulable protein, and the experiment must be repeated 

 with urine diluted to a smaller extent. If the ring appears after a 

 shorter interval than three minutes, the diluted urine contains more 

 than i part in 30,000 (the original urine more than 0*033 P er cent.) 

 and must be further diluted. Fill a burette with the diluted urine. 

 Run i c.c. of it into a test-tube and add 9 c.c. of distilled water. 

 Repeat the test with this second dilution. If the ring appears at a 

 longer interval than three minutes, the twice-diluted urine contains 

 less than i part of albumin in 30,000, and the original undiluted 

 urine less than i part in 300 i.e., less than 0*33 per cent. So far, 

 then, we have found, let us suppose, that the proportion of albumin 

 in the original urine lies between 0*033 and 0*33 per cent. Now run 

 i c.c. of the urine of the first dilution (the urine diluted ten times) 

 into a test-tube, and add 4 c.c. of distilled water i.e., dilute again five 

 times. If this gives the white ring in Heller's test in three minutes, 



the original urine will contain i part of albumin in -* i.e., in 



10 x 5 



