SERUM DIAGNOSIS OF SYPHILIS. 165 



c.c. are needed +0.4 c.c. for the antigen control tube = 1.9 c.c. in all or 2.0 c.c. in round 

 numbers. This amount is diluted with normal salt solution in the proportion of 1 15 so 

 that 8 c.c. of saline are added (=2:10), i c.c. of this dilution contains 0.2 antigen and 

 1/2 c.c. contains o.i antigen. The first tube (i, 4, 7, 10, etc., in diagram) of every test 

 therefore receives i c.c., the second tube 1/2 c.c., the third tube nothing, the antigen tube 

 (tube 19) 2 c.c. Physiological salt solution is added to make up i c.c. in each tube; 

 first tube nothing; second tube 1/2 c.c.; third tube i c.c. of saline. 



The normal extract required for the tubes in the second rack is similarly estimated, 

 0.2 c.c. is needed for each test, 5 (tests) X 0.2= 1.0+0.4 for the antigen control tubes 

 = 1.4 or 1.5 c.c. in round numbers. For purposes of dilution 1 15, 6 c.c. of salt solution 

 are added and i c.c. ( = 0.2 of extract) placed into each of the tubes (20 to 26) and 2 c.c. 

 into the normal extract control test-tube (tube 27). In this series also, salt solution is 

 added to make up equal quantities of i c.c.: first tube nothing, second tube 1/2 c.c., third 

 tube i c.c.; antigen tube 27, nothing; system (28), complement (29) and blood (30) 

 control tubes each i c.c. 



The second ingredient of the test is next added, i.e., the respective serum. This is 

 not diluted but added directly; 0.2 c.c. into the first tube of each test; o.i c.c. into the 

 second; 0.2 c.c. into the third tube; also 0.2 into the control series of tubes labelled with 

 the patient's names in the second rack. Salt solution is again added to make up to the 

 equal quantity of 2 c.c. in each tube, thus: 0.8 c.c. into first, o. 9 c.c. into the second, o. 8 

 c.c. into the third tube, and 0.8 c.c. into the control series; nothing into the antigen tubes, 

 i c.c. into system, complement, and blood control tubes. 



The addition of complement follows next. Each tube, except the blood tube, receives 

 o.i of complement. Thus the tubes are counted and if, for example, ninteen tubes are 

 present 19X0.1 c.c. complement is taken, or in round numbers 2 c.c. 



Complement is always diluted i : 10, or 2 c.c. complement + 18 c.c. saline, so 

 that each tube except the blood tube (30) receives i c.c. of this diluted complement 

 Tube (30) receives i c.c. of saline instead. All tubes are then carefully shaken and 

 the racks placed into the incubator for one hour. 



During this time, the hemolysin and washed red blood cells are properly diluted. 

 The red blood cells are made up in a 5 per cent, suspension of which each tube will 

 receive i c.c. Thus in the present test thirty tubes exist, requiring 30 c.c. of blood 

 suspension; since i c.c. of washed blood when diluted i: 20 will supply twenty tubes, 

 for 30 c.c. about i 1/2 c.c. of blood will be required, or 2 c.c. will make 40 c.c. of a 

 5 per cent, blood suspension. 



As for the hemolysin, its titer for example is i : 2000 and it is employed in the dilution 

 of i : 1000. Each tube except the blood and complement tubes will receive i c.c. 

 of the diluted hemolysin; i c.c. of the latter if diluted properly would give 1000 c.c.; 

 o . i of the hemolysin which is the smallest amount that can be measured out, will give 

 100 c.c. Every tube except 28 to 30 will receive i c.c. of the hemolysin dilution i : 1000. 

 Tubes 29 and 30 will receive none (replaced by saline), tube 28 will receive 1/2 c.c. of 

 this hemolysin and 1/2 c.c. of saline. 



After an hour's incubation, each tube receives i c.c. of R. B. C. and i c.c. of the 

 hemolysin just mentioned. If it is desired to hasten the results, it is advisable to mix a 

 sufficient equal quantity of R. B. C. and hemolysin solution (30 c.c. of each) and allow 

 the mixture to remain in the incubator for a short time before the hour's incubation 



1 0.4 c.c. of luetic serum frequently binds complement of its own accord. Experience has 

 shown that if 0.2 c.c. does not bind complement and 0.2 c.c. of serum + 0.2 c.c. of antigen does 

 bind complement, the unknown serum is surely of luetic origin. 



