362 MARSHALL AND TEAGUE. 



It is generally customary among all authorities to use I cubic centimeter 

 of a 0.1 dilution of complement as a standard dose. 



The total volume of fluid employed by different authorities varies from 2.5 

 to 10 cubic centimeters. 



b. Time and temperature. — The general advice given by Wassermann with 

 regard to time and temperature is to allow the antigen, antibody and com- 

 plement to remain in contact for one hour at 37°, subsequently allowing this 

 mixture to act upon the ho?molytic complex for two hours at 37°. Some author- 

 ities allow antibody, antigen and complement to remain at room temperature. 

 The result may be observed upon removing the test tubes from the incubator, 

 or they may be placed on ice and the result noted on the following morning. 



Meier (3) thinks it is very important to observe closely the experiment in the 

 incubator and not t let it run automatically for two hours. As soon as lysis is 

 complete in all of the controls, remove the test tubes from the incubator and put 

 them on ice without waiting for the expiration of two hours. Frequently from 

 three-quarters to one hour suffices, especially if the corpuscles and hemolytic 

 amboceptor have been previously mixed for one-quarter to one-half an hour. 

 This is a great improvement, and does away to a large extent with the "Nachlos- 

 ung" or haemolysis which develops in some test tubes after being removed from 

 the incubator and placed upon ice. It is even better to bind hemolytic amboceptor 

 to the corpuscles by allowing them to stand in contact for one-half hour, and 

 remove the excess of serum by centrifugation and suspend the laden corpuscles 

 in fresh salt solution. 



c. Controls. — The controls which are required in conducting this experiment 

 are numerous. In addition to the preliminary tests mentioned above, each experi- 

 ment must be accompanied by the following controls. 



A parallel series of tests must be made — 



1. With the serum under examination and normal extract. In these controls 

 there should be no deflection; if deflection occurs, it is not specific and the experi- 

 ment must be thrown out. 



2. With standard fresh syphilitic serum and the extract of syphilitic liver. 

 In this control there should be definite deflection, otherwise the extract is defective, 

 and the experiment fails. 



3. With standard fresh syphilitic serum and extract of normal liver. In this 

 control there should be no deflection. If it occurs either the standard. serum is 

 deteriorating or the total quantity of colloids is sufficient to produce a non-specific 

 deflection. 



4. With serum which is certainly not syphilitic and the luetic extract. In this 

 control there should be no deflection. If it occurs the experiment is valueless and 

 a fresh luetic extract must be prepared. 



5. With the antibody serum and complement alone. This test should give no 

 deflection. 



6. With the luetic extract and complement alone. This test should give no 

 deflection. 



7. With the red corpuscles. hemolytic amboceptor and complement, which has 

 been previously heated to 37° for one hour. In this control there should be 

 complete haemolysis. 



8. With blood corpuscles in salt solution alone. 



9. With blood corpuscles and hsemolytic amboceptor alone. 



10. With blood corpuscles and complement alone. 



11. With blood corpuscles and organ extract alone. In these controls there 

 should be no haemolysis. 



Meier (3) remarks that as experience increases, it becomes less necessary to 

 make daily controls of the standard luetic serum with extracts and of serums 



