644 APPENDIX: LABORATORY METHODS 



with accurately calibrated pipettes into wide agglutination tubes (di- 

 mensions of 10 by 75 mm. have been found suitable). If 0.5 cc. of se- 

 rum has been used, 1 cc. of saline solution is then added to each tube, 

 followed by 0.5 cc. of a saline solution containing 1 mg. of the specific 

 polysaccharide per cubic centimeter, to make a total volume of 2 cc. 

 Ordinary uncalibrated pipettes are adequate for the saline and poly- 

 saccharide solutions. The tubes are plugged and the contents carefully 

 and thoroughly mixed by a rotary motion imparted by drawing the fin- 

 ger tips rapidly and repeatedly in a diagonal stroke down the side of 

 the tube. The tubes are allowed to stand 2 hours in the water-bath at 

 37° and overnight in the ice-box.* The plugs are then removed and the 

 tubes centrifuged for 10 minutes at 1,000 r.p.m., either in a refrigerat- 

 ing centrifuge or immersed in ice-water. The supernatant liquid is then 

 carefully drained off by inverting the tube and wiping the mouth of the 

 tube with filter paper. The tubes are then placed in ice-water and each 

 precipitate is washed with 2 cc. of an ice-cold, 1 to 20,000 saline solu- 

 tion of the specific polysaccharide, mixing the contents as before. It ap- 

 pears to make little difference whether the disc of precipitate is loos- 

 ened from the bottom of the tube. After one-half hour in the cold, the 

 tubes are again centrifuged and drained as before. About 0.5 cc. of 

 water is then added to each tube, with shaking until the precipitate is 

 loosened, after which the disc is dissolved by the addition of 2 drops of 

 normal sodium hydroxide solution with rotation of the tube until the 

 precipitate has disappeared. If the disc is allowed to stick to the glass, 

 solution is much slower. The solution is then rinsed quantitatively into a 

 micro-Kjeldahl flask or tube and the nitrogen determined by any stand- 

 ard procedure. The Pregl method, slightly modified, is satisfactory. 

 Nitrogen found multiplied by 6.25 equals specifically precipitated pro- 

 tein. 



c. Modified routine] (Barnes, Clarke, and Wight 82 ). The test is con- 

 ducted by mixing a constant amount of capsular polysaccharide with 

 varying dilutions of immune serum and comparing the precipitin titer 

 of the unknown serum with that of a control serum tested simultane- 

 ously. 



(1) Dilution of SSS: Stock solutions of SSS are made up in 1 to 



* Sterile technique should be employed up to this point. In the case of low- 

 grade serums, identical values are obtained if the tubes are allowed to stand in 

 the water-bath only one-half hour, followed by one-half hour in ice-water before 

 proceeding. With more potent serums, results a few tenths of a milligram per 

 cubic centimeter too low are obtained by shortening the process in this way. 



t Suitable only for unconcentrated serums. 



