RUTH GILBERT 841 



0.2, 0.1, 0.05, 0.025, 3.nd o.oi cc. are tested. Even smaller amounts are employed if inhibition 

 of hemolysis still occurs. 



Three-quarters of an hour at room temperature followed by three-quarters of an hour at 

 37° C. are allowed for fixation. 



A 5 per cent suspension of sheep cells with two and one-half units of amboceptor con- 

 tained in 2 cc. are used in the hemolytic system. 



As complement, guinea pig serum, titrated in a i : 10 dilution both with and without 

 antigen, is used. The unit is determined after two hours in the incubator at 37° C. The de- 

 termination without antigen is made for use in the serum control and the one with antigen 

 for use in the test proper. 



Extracts of human heart and syphilitic liver are used as antigens. 



After the addition of the hemolytic system, the tests are incubated for two hours at 

 37° C. and a hemoglobin scale is used for reading the results. 



METHOD USED BY MULLER, SERO-DIAGNOSTIC LABORATORY, VIENNA' 



The patient's serum is inactivated, and four drops are used in the test, a drop correspond- 

 ing to about 0.05 cc. 



One drop of guinea pig serum is used as complement. This must be one and one-half 

 times the amount necessary for hemolysis. A unique method of diluting the antigen is 

 recommended. To 20 cc. of an alcoholic extract of beef heart tissue is added 4 cc. of a 0.5 

 par cent solution of cholesterin and the mixture evaporated to 8 cc. This is poured quickly 

 into a beaker 7 cm. in diameter which contains 5 cc. of 0.9 per cent sodium chloride. Next, 

 20 cc. of salt solution which has been kept in a second beaker are added as quickly as possible. 

 This mixture must mature at 56° C. for from twelve to twenty-four hours. Five drops are 

 used in the test. 



One hour in the incubator is allowed for fixation. 



The results of the tests are recorded after hemolysis has occurred in most of the tubes 

 containing normal sera, and a final record is made one hour later. 



In addition to the usual controls, one control is recommended in which alcohol is used 

 instead of antigen, to insure "that fixation will not occur with the amount of alcohol used in 

 the antigen. The test differs from many others in that 2.5 cc. of salt solution are put in each 

 tube before the other reagents, which are, with the exception of antigen, added undiluted by 

 dropping them from a pipette. 



METHOD USED BY MUTERMILCH IN THE PASTEUR INSTITUTE, PARIS^ 



The procedure of Bauer-Hecht, modified by Levaditi and Latapie, Weinberg, Muter- 

 milch, and Latapie, is employed. 



Human serum that has not been inactivated is tested in o.i-cc. amounts. 



The complement and antisheep amboceptor in the patient's serum which is being tested 

 is used. The hemolytic index for each serum is determined according to Weinberg's pro- 

 cedure. One-tenth of a cubic centimeter of each serum is tested with amounts of the 5 per 

 cent suspension of sheep cells varying from o.i through i cc. If the sera contain unusuall\- 

 large amounts of amboceptor, they are tested with amounts of the cell suspension varying 

 from I to 2 cc. These tubes are incubated for one hour at 37° C. In the case of spinal fluids 

 or sera not having natural complement or antisheep amboceptor, active human serum from 



• Ibid. 



' "La Technique du sero-diagnostic de la syphilis actuellement employee a ITnstitut Pasteur 

 k Paris," Ann. de Vlnst. Pasteur, 38, 827. 1924. 



