A STUDY OF FIXATION REACTIONS. 361 



normal organs. Wassermann and Plaut(S) note that the aqueous extract under- 

 goes sudden and rapid alterations in deflecting power. This is confirmed by 

 others including Morgenroth and Stertz. 



D. FINAL PRECAUTIONS. 



In a preliminary test determine that the suspected serum does not cause 

 deflection with normal extract. If there is enough serum available, test its 

 action upon sheep's corpuscles with complement. Human and monkey serum, 

 normal or luetic is often quite strongly hsemolytic for sheep's corpuscles, at 

 times 0.1 cubic centimeter producing haemolysis of 1.0 cubic centimeter of a 5 

 per cent suspension. 



The test tubes, glassware and other apparatus must be irreproachably clean, 

 the extracts and serums must be collected and preserved under aseptic precautions, 

 and the experiments must be conducted in such a way as to avoid contamination 

 by bacteria, dust, etc. 



3. Details of the Actual Experiment. 



a. Amounts of materials. — Wassermann, Neisser, Bruck, and Schueht(2) use 

 0.1 cubic centimeter of each one of the ingredients, bringing the total volume in 

 each test tube up to 5 cubic centimeters with normal salt solution. Meier(3) 

 dilutes his materials so that he needs only to measure out 1 cubic centimeter of 

 each ingredient, making the volume constant with salt solution. He uses two 

 quantities, 0.2 and 0.1 of the antibody and also of the antigen. Michaelis(l) 

 and others also use 0.2 or 0.1 of serum from the suspected case with 0.1 or 0.2 

 of extract of syphilitic organs. Landsteiner and Stankovic(9) use 10 parts of 

 0.8 per cent salt solution; 1 part of serum inactivated at 56° for one-half hour; 

 1 or 2 parts of extract and 1 part of guinea-pig complement. After one hour 

 at 37° they add 1 part of 50 per cent sheep's corpuscles suspension free from 

 serum and previously treated with two dissolving doses of hsemolytic serum, 

 incubate for one hour and a half at 37°. 



Morgenroth and StertzO) recommend wider variations in the amounts of sub- 

 stances employed, especially of the antigen. They show that the zone within 

 which deflection occurs is narrow, in one experiment cited deflection occurring 

 when 0.001 to 0.0025 of antigen was employed, but failing to occur with greater 

 or less amounts. 



In most of the literature, 0.1 cubic centimeter of the extract of antigen was 

 employed in each test tube. Meier(3) finds that this usually suffices when mixed 

 with 0.1 cubic centimeter of the luetic serum, while it produces no deflection 

 with 0.2 cubic centimeter of normal serum. If the extract of antigen is weaker 

 he uses from 0.2 to 0.4 cubic centimeters with 0.1 cubic centimeter to 0.2 cubic 

 centimeter of the luetic serum, always making a control to prove that the same 

 dose of extract produces no deflection with even twice the dose of normal serum. 



Meier(3) notes that the antibody varies quantitatively in different cases and 

 the quantitative test is necessary for exact scientific work. In practice, however, 

 it is necessary to use only two dilutions, neither of which is so strong as a 

 control with normal serum need be in order that the normal serum produces 

 deflection. In practice Meier uses a dilution of one in five of antibody, that 

 is 0.2 culjic centimeter for one quantity and for the other one-half of this, viz, 

 one part in ten, or 0.1 cubic centimeter of antibody, but in a few of his cases 

 0.005 cubic centimeter (=1: 200) of serum, deflected completely when mixed with 

 luetic extract, while even 20 times the amount (0:1 cubic centimeter) gave 

 complete lysis, i. e.. no deflection, when mixed with extract of normal organs. 



Michaelis and Lesser(4) also employ about 0.2 cubic centimeter of serum 

 from the patient. 



