480 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



0.1, 0.2, 0.3, 0.4, 0.5 c.c. To each tube add four drops (0.08 c.c.) of 

 inactivated or one drop (0.02 c.c.) of fresh active syphilitic serum; also 

 0.1 c.c. (five capillary drops) of complement (40 per cent.) and 1 c.c. of 

 1 per cent, corpuscle suspension. Then add sufficient salt solution to 

 bring the total up to 2 c.c. 



Two controls should be included: (1) The serum control, containing 

 the dose of serum, 0.1 c.c. of the complement, 1 c.c. of corpuscle sus- 

 pension, and saline solution; (2) the hemolytic control, containing at 

 this time 0.1 c.c. of complement, 1 c.c. of corpuscle suspension, and 

 sufficient saline solution. 



All tubes are incubated for one hour at 37 C. (one-half hour in 

 water-bath), after which two units of amboceptor are added to each 

 tube. The tubes are then shaken gently and reincubated for two hours 

 (one hour in water-bath). At the end of this time the two controls 

 should be completely hemolyzed, and in the series proper that tube 

 showing just complete inhibition of hemolysis contains one antigenic 

 unit. Usually the first and second tubes show some inhibition of 

 hemolysis, and in the third and other tubes hemolysis is completely 

 inhibited. In this case 0.2 c.c. of this emulsion would be one anti- 

 genic unit ( = 0.02 c.c. undiluted antigen): five times this amount equals 

 0.1 c.c. of the first emulsion (1:10), which is the amount to be used in 

 making the main tests. 



Unless the antigen shows signs of deterioration, these titrations need 

 be made only about once a month. 



If paper antigen is employed, both titrations are conducted in 

 exactly the same manner by adding increasing lengths of a strip of 

 dried paper 5 mm. in width, impregnated with the antigen. 



5. Fluid to be Tested. If active serum is used, it should be fresh, 

 free from hemoglobin, and preferably not over twenty-four hours old. 

 The dose is 0.02 c.c., or one capillary drop; inactivated serums are used 

 in doses of 0.08 c.c., or four capillary drops. Cerebrospinal fluid is used 

 unheated in doses of 0.2 c.c., or 10 capillary drops. Sufficient blood 

 for this test may be collected in a Wright capsule. (See p. 33.) 



6. The Test. The complement, amboceptor, antigen, and serums 

 may be conveniently measured by drops from a capillary pipet (Fig. 

 2). In placing a drop the pipet should be held uniformly at an angle 

 of 45 degrees, or else the size of the drop will differ, depending on whether 



t the pipet is held vertically or horizontally. 



Arrange four pairs of small test-tubes (10 by 1 cm.) in a rack con- 

 taining two rows cf holes. Into each of the tubes on the front row 



