REFRACTOMETRIC METHOD 61 



ishing the tendency to crack on cooling. Into one of these tubes, 

 which, has been carefully cleaned and dried, is now introduced 

 exactly 1 cc. of serum with the aid of an accurately calibrated 

 pipette with a capillary tip. Such pipettes may be prepared by 

 taking lengths of narrow-bore glass tubing drawn out to a capil- 

 lary at one end, introducing 1 cc. of mercury, and marking with 

 a diamond the extremities of the mercury column. The mercury 

 is then delivered into another similar tube, which is similarly 

 marked, and the operation repeated until the desired number of 

 pipettes is obtained. Prepared in this way the pipettes all deliver 

 (between the marks) the same volume of fluid, and if this is the 

 case the exact volume employed is immaterial provided it is in 

 the neighborhood of 1 cc. 



In delivering the serum, wetting the upper part of the tube and 

 the formation of air-bubbles should be carefully avoided. 



The serum having been introduced, with another pipette cali- 

 brated against the first, deliver 1 cc. of N/25 acetic acid solution, 

 which may be made up with sufficient accuracy by diluting 4 cc. 

 of glacial acetic acid to 1750 cc. 



A short length of thick platinum, silver, or nickel wire is now 

 introduced into the tube and the upper end is sealed off in a flame, 

 taking care not to heat the contents. After cooling, the tube is 

 shaken and the short length of wire contained in it brings about 

 a thorough admixture of the contents. The tube is now placed 

 in a beaker of cold water of such a depth as to immerse the top 

 of the contained column of liquid several millimeters below the 

 surface. It is well to rest the bottom of the tube upon a wad of 

 glass wool or a piece of wire gauze to avoid the cracking of the 

 tube by bumping during the subsequent boiling. The water is 

 now slowly heated to boiling and allowed to boil energetically 

 for exactly two minutes. The tube is then removed from the 

 boiling water and allowed to cool to room temperature. 



When cool the tube is broken open a little above the surface 

 of its contents and the coagulum is broken up. This is best 

 accomplished with the aid of a platinum wire about 0.6-0.7 mm. 

 in diameter and provided with several slight bends. This is 

 inserted into the tube and the upper end twirled between the 

 thumb and forefinger. The fluid and the coagulum are now 

 separated by centrifugalization, the fluid is withdrawn by the 

 aid of a dry, clean pipette, and the refractive indices of the fluid 



