REFRACTOMETRIC METHOD 63 



short pieces of rubber tubing, are employed as stoppers. Into 

 one of these tubes is introduced, with the aid of a pipette similar 

 to that described above, 0.5 cc. of a saturated solution of ammo- 

 nium sulphate (prepared by dissolving an excess of ammonium 

 sulphate in hot water, allowing the excess to crystallize out on 

 cooling and then removing the fluid from the subnatant crystals 

 and keeping in a well-stoppered container). With another pi- 

 pette which has been calibrated against the first, introduce 0.5 cc. 

 of serum, drop in a piece of platinum, silver or nickel wire, affix 

 the stopper and shake thoroughly with as little delay as possible. 

 It is necessary to introduce the ammonium sulphate first, as other- 

 wise, being of greater specific gravity than the serum, it sinks 

 through the serum, portions of which are thus exposed for some 

 time to ammonium sulphate of higher concentration than one- 

 half saturated. This leads to a precipitation of albumins which 

 do not readily redissolve, and the results obtained are erroneous 

 and irregular. If the ammonium sulphate is introduced first 

 the serum floats on the top of it and energetic shaking brings 

 about almost immediate admixture of the serum and the reagent. 

 It is well, while shaking, to hold the thumb against the bottom 

 of the tube, thus diminishing the danger of cracking the tube 

 by the impacts of the heavy piece of wire. 



The tube, with the stopper still affixed, is now centrifuged. 

 The precipitate soon settles and sufficient supernatant fluid may 

 ,be drawn off to fill the tip of a pipette and the space between 

 two marks known to hold about 0.25 cc. (prepared as above). 

 This quantity of the supernatant fluid is delivered into another 

 clean, dry tube of the type employed in the precipitation, and 

 0.25 cc. of distilled water is added with the aid of a pipette cali- 

 brated against the first. A piece of wire is dropped in, a clean 

 stopper affixed, and the contents are shaken. We now deter- 

 mine the refractive index of this fluid and that of one-quarter 

 saturated ammonium sulphate solution prepared (and kept as a 

 stock solution) by mixing equal volumes of saturated ammonium 

 sulphate solution and distilled water and adding to this mixture 

 an equal volume of distilled water. The difference between these 

 refractive indices represents one-fourth of the combined refrac- 

 tivities of the albumins and of the non-protein constituents. 

 Multiplying by 4, therefore, and subtracting the refractivity 

 of the non-protein constituents, we have the refractivity of the 



