NEPHELOMETRIC METHOD 59 



The coagulating reagent employed by Kober is sulpho- 

 salicylic acid. The following are the details of the method 

 as applied by Kober to the estimation of the various proteins in 

 milk (8). 



Five cubic centimetres of milk are carefully measured into a 

 250-cc. graduated flask and after adding 200 cc. of distilled water 

 and 10 cc. of decinormal sodium hydroxide solution, water is 

 added to the mark and the solution is thoroughly shaken. Ten 

 cubic centimetres of this mixture is then placed, together with 

 2 cc. of ether (which has previously been washed with a 10 per 

 cent aqueous solution of sodium hydroxide) in a centrifuge tube 

 which is then tightly stoppered with a cork and vigorously shaken. 

 After allowing the mixture to stand until the layers have sepa- 

 rated, or after centrifuging for one to two minutes, the cork is 

 removed and 5 cc. of the aqueous layer is withdrawn. This is 

 done by closing the top of the pipette with the finger and insert- 

 ing it quickly into the centrifuge tube. If this is done correctly 

 the ether solution will not contaminate the sample. This 5 cc. 

 of the aqueous layer is then diluted in a volumetric flask to ex- 

 actly 50 cc. 



The milk treated in this way has an almost inappreciable tur- 

 bidity, not more than 1.6 per cent of the turbidity which is sub- 

 sequently produced by the coagulating agent. Such residual 

 turbidity as it possesses is almost exactly equal to that of the 

 standard solution of casein with which it is subsequently com- 

 pared, and is therefore without influence upon the accuracy of 

 the comparison with the standard. 



To 10 cc. of this solution is now added 10 cc. of a 3 per cent 

 solution of sulphosalicylic acid and the suspension of coagulated 

 milk protein which is thus obtained is matched in the nephel- 

 ometer with the following standard: One volume (5 cc.) of a 

 0.01 per cent casein solution to which is added two volumes 

 (10 cc.) of 3 per cent sulphosalicylic acid. 



The volume of solution employed is not a correct aliquot of 

 the original sample of milk, owing to the fact that in the extrac- 

 tion with ether some ether is dissolved by the aqueous layer and 

 some water by the ether layer. A determination of the volume 

 increase of a solution of sodium caseinate shaken up with ether 

 shows that 10 cc. of the clear aqueous layer of defatted diluted 

 milk is equal to 9.1 cc. of the diluted milk before extraction with 



