642 APPENDIX: LABORATORY METHODS 



Precipitation 



There are various methods of titrating antipneuraococcic serum based 

 on precipitation of the antibody by the specific polysaccharide. Those 

 listed below are given to illustrate the methods in more common use. 



a. Optimal proportions (Smith 1298 ). The test is carried out in three 

 routine stages. 



(1) Preliminary Orientation: The object is to decide upon a suit- 

 able dilution in which to use the serum in the subsequent tests, for if 

 the serum is used insufficiently diluted, precipitation is so rapid that 

 it becomes impossible to decide which tube is the optimal one. A suit- 

 able serum dilution is that which produces macroscopic precipitation 

 with the equivalent amount of antigen in 20 to 30 minutes. Dilutions 

 of serum, 1 in 5, 1 in 10, 1 in 20, are made with 0.85 per cent salt 

 solution and 1 cc. of each mixed with 1 cc. of polysaccharide in 1 in 

 50,000 dilution. The tubes are placed in the water-bath at 40° and 

 are examined at the end of 5 minutes and again at 10 minutes. The 

 choice of the serum dilution to be used in the subsequent tests, based 

 on the result obtained, is a matter of personal judgment, readily ac- 

 quired from a few experiments. 



(2) Rough Test with Widely Spaced Antigen Dilutions: A row of 

 eight tubes is numbered from left to right and 1 cc. of buffer solu- 

 tion (borate buffer solution, pH 8.0) is run into each. To the first 

 tube, 1 cc. of a stock solution of SSS, 1 in 10,000 dilution, is added 

 by means of an accurate 1 cc. pipette, the contents are mixed thor- 

 oughly and 1 cc. of the mixture is carried over to tube 2, and so on, 

 1 cc. being discarded from the last tube. It is not necessary to use 

 separate pipettes. To each tube in turn, 1 cc. of the chosen dilution 

 of serum is added by means of a mounted syringe, care being taken 

 to prevent the stream of serum from striking the walls of the tube. 

 (A 2 cc. all-glass syringe is mounted vertically on a rigid stand with 

 an adjustable screw over it, so that when the piston is raised to meet 

 the screw, the syringe capacity is exactly 1 cc. A 23-gauge half-inch 

 needle is fitted to the syringe, which can now be filled rapidly and 

 accurately with serum, held in a conical centrifuge tube.) The rack of 

 tubes is placed in the water-bath and transferred periodically for a 

 rapid examination to a reading box, so designed that the whole series 

 of mixtures, evenly transilluminated, can be viewed against a dead 

 black background. 



In this rough test, one tube usually shows precipitation long before 



