COMPOSITION OF HUMAN MILK IN AUSTRALIA. 177 



as in Sikes' method, but was found later not to reduce the 

 ash-content of the precipitate of protein. The percentage 

 of ash in the precipitates was found to be about five ; 

 Sikes found the percentage of protein to be about three in 

 his precipitates, but in the present case the percentage of 

 ash in the precipitates obtained by his method was the 

 same as in those obtained by the method described. 



The aqueous layer of the liquids obtained in the estima- 

 tion of fat by the Rose-Gottlieb method is not clear, but 

 opalescent, owing to the presence of substances in colloidal 

 solution, the chief of which are caseinogen and phosphates. 

 On standing, part of this fine suspension slowly rises and 

 collects beneath the ethereal layer. When this has 

 occurred, the distribution of the substances in the aqueous 

 layer is no longer uniform, so that, in taking an aliquot 

 part of this layer for the estimation of protein, a represen- 

 tative sample might not be obtained, and a fallacious result 

 might be arrived at, although, as the precipitate is probably 

 composed of phosphates which have been thrown down by 

 the addition of ammonia, the effect of this uneven distri- 

 bution of the contents of the aqueous layer upon the amount 

 of protein found would probably be insignificant. The 

 liquids, however, should not be allowed to stand longer 

 than about one hour, or should be shaken up and allowed 

 to settle again if they have stood too long. 



The following protocol of an estimation of fat and protein 

 on the same sample of milk will give a more definite idea 

 of the method outlined above. 



Ten cc. of human milk in a 100 cc. stoppered measuring cylinder 

 were mixed with 2 cc. of 10% ammonium hydroxide and 10 cc. of 

 95% methylated spirit; 25 cc. portions of ether and of petroleum 

 spirit were then added, the mixture being thoroughly shaken after 

 each addition, and finally allowed to stand for about one hour. 

 The levels of the ethereal and aqueous layers were then found to 

 be at 70- 6 cc. and 19'0 cc, respectively. The volume of the 



K— August 4, 1915. 



