338 PHYSIOLOGICAL CHEMISTRY 



cow's milk into another similar tube. Add to the contents of each tube i drop of 

 a 0.25 per cent aqueous solution of nile-blue sulphate (Griibler). Shake the 

 tubes gently and permit them to stand undisturbed for 10-30 minutes. The 

 milk assumes a bluish cast in each case. At the end of the lo-minute interval 

 add 10 c.c. of ether to the contents of each tube and shake very thoroughly for one 

 minute. The ether extracts the pigment from the human milk, leaving the milk 

 white. In the case of cow's milk the ether does not extract the dye and the milk 

 remains bluish in color. 



11. Tests to Differentiate between Raw Milk and Heated Milk. 



(a) Tricresol Peroxidase Reaction (Kastle) . The peroxidase reaction of 

 milk is founded upon the fact that small amounts of raw milk will in- 

 duce the oxidation of various leuco compounds by hydrogen peroxide. 

 This reaction has been used in a practical way as the most convenient 

 means of differ en tia ting between raw milk and heated milk. Many 

 substances have been employed for this purpose, e.g., guaiac, para- 

 phenylenediamine, ortol, amidol, etc. Kastle has found that a dilute 

 solution of "tricresol" 1 acts as a sensitizing agent in the peroxidase 

 reaction and offers the following test which is based upon this fact. 



Procedure. To 2-5 c.c. of raw milk in a test-tube add 0.1-0.3 c.c. of M/io 

 hydrogen peroxide and i c.c. of a i per cent solution of "tricresol." A slight 

 though unmistakable yellow color will be observed to develop throughout the 

 solution. Repeat the test using milk which has been boiled or heated to 

 8oC. for 10-20 minutes and cooled, and note that no yellow color is produced. 



The color reaction in the case of the raw milk probably results from 

 the oxidation of the cresols by the hydrogen peroxide. The first 

 product of this oxidation 2 then oxidizes the leuco compound, when such 

 is present, and causes the color observed. 



(6) Benzidine Peroxidase Reaction (Wilkinson and Peters}. 3 To 10 c.c. of the 

 milk to be tested add 2 c.c. of a 4 per cent alcoholic solution of benzidine, suffi- 

 cient acetic acid to coagulate the milk (usually 2-3 drops) and finally 2 c.c. of a 

 3 per cent solution of hydrogen peroxide. Raw milk yields an immediate blue 

 color. In adding the peroxide it is best to permit it to flow slowly down the 

 wall of the vessel containing the mixture instead of allowing it to mix with the 

 milk. Milk which has been heated to 78C. or above remains unchanged. 



The reduction of the CO 2 content of milk through heating has also been sug- 

 gested as a means of differentiating between raw and heated milk. 4 



12. Saturation with Magnesium Sulphate. Place about 5 c.c. of milk in a 

 test-tube and saturate with solid magnesium sulphate. What is this precipitate? 



13. Influence of Gastric Rennin on Milk. Prepare a series of five tubes as 

 follows : 



1 "Trikresol" is the trade name of an antiseptic which contains the three cresols in ap- 

 proximately equal proportions. 



2 Probably some organic peroxide or quinoid compound. 



3 Wilkinson and Peters: Z. Nahr-Genussm., 16, No. 3, p. 172. 



4 Van Slyke and Keeler: Jour. Biol. Chem., 42, 41, 1920. 



