PRACTICAL METHODS IN IMMUNITY 



9. At the expiration of 30 minutes from the commencement of incubation for 

 complement binding, add i volume of haemolytic system to each of the tubes, I, II, 

 III, etc., in the order of antigen dilution. 



10. Finally, after washing pipette in salt soultion, add i volume of haemolytic 

 system to control in tube X. (If the haemolytic system should be added to the con- 

 trol tube before the antigen tubes, complement from the control tube might be 

 carried over to the antigen tubes.) 



FIG. 49. i. Copper water bath 12X12X8 inches, (a) Thermometer to show 

 38 C. (b) Tubes containing antigen dilutions, (c) Tube containing hamolytic 

 system incubating along with the antigen tubes. 2. Ordinary rice cooker with copper 

 holder for test-tubes. 



Shake each tube thoroughly. Allow them to incubate for a few minutes. Then 

 examine tubes I, II, III, etc., for haemolysis. The control should, of course, show 

 haemolysis. The antigen tubes should show a white, supernatant fluid over the in- 

 tact red cell sediment in the tubes with the low dilutions and even in the highest 

 dilutions, where the serum is strongly positive. In a weakly positive serum, in- 

 hibition of haemolysis may only show in the first two tubes and haemolysis show in 

 those tubes having higher dilutions of antigen. 



It will be noted that the reagents are made in accordance with 

 Noguchi's directions. Even in those cases where fresh guinea-pig 

 serum is employed to replace complement, absent from the human 

 serum tested, we employ the 40 % solutions used in Noguchi's tech- 



