VI. SEROLOGICAL REACTIONS 645 



1,000 dilution with borate buffer solution (pH 8.0). From this stock 

 solution, various dilutions are tested against varying dilutions of a 

 control serum to determine the suitable dilution of the carbohydrate 

 to be used routinely. This dilution will vary from lot to lot and ac- 

 cording to the type of SSS. To illustrate, for Type I, the dilution 1 

 to 30,000; for Type II, 1 to 80,000; for Type V, 1 to 50,000; and 

 for Type VIII, 1 to 80,000 may be found to be satisfactory. 



(2) Dilution of Serums: Serums are diluted with physiological 

 salt solution in series, 1 to 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 

 120, 160, 200, 240, 320, 400, and so on. For the test, ten adjacent 

 dilutions from the series are employed. 



(3) The Test: 0.5 cc. of the dilution of SSS chosen is added to 0.5 

 cc. of the serum dilutions, and the contents mixed by shaking the 

 racks for several minutes. The mixtures are incubated for two hours 

 at 40° and overnight in the refrigerator at 4°. 



(4) Interpretation: The end point is taken as the highest dilution 

 of serum showing definite precipitation. By comparing the titer of 

 the unknown serum with that of the control, the number of units in 

 one cubic centimeter of the unknown serum can be calculated. 



d. Combining equivalents (Felton and Stahl 433 ). 



(1) Preparation of SSS Dilutions: The stock solution is made in a 

 volumetric flask from an accurately weighed sample of the dried ma- 

 terial, dissolving it first in a small volume of saline solution with the 

 addition of sufficient normal NaOH to maintain a neutral reaction 

 and facilitate solution. Then salt solution is added to complete the 

 volume for a 1 to 500 dilution. Using a volumetric flask and a quan- 

 titative delivery pipette, the dilution required for the test is pre- 

 pared in such a quantity that not less than 1 cc. of the 1 to 500 stock 

 solution need be measured. 



(2) Preparation of Serum Dilutions: Physiological salt solution 

 for the serum dilutions is measured with a burette when more than 2 

 cc. is required, and with a 2 cc. accurately calibrated serological 

 pipette, graduated in tenths, when less than 2 cc. is needed. Serum 

 for the initial dilution is drawn up to the line in a quantitative pi- 

 pette calibrated to contain exactly the amount required. Excess serum 

 clinging to the outside of the pipette is removed with a piece of cot- 

 ton or gauze, and the pipette is rinsed five times in the dilution be- 

 ing made. Two cc. serological pipettes graduated in tenths are used 

 in making subsequent dilutions. Each dilution should be in at least 1 

 cc, and preferably 2 cc. or more, volumes. 



